Long wavelength fluorogenic intracellular ion indicators that are well retained in the cytosol

ABSTRACT

Cell permeable metal ion indicator compounds and methods of their use and synthesis are described. The compound comprises a metal chelating moiety (M c ), a reporter molecule and two or more lipophilic groups (G L ) covalently bonded through a linker to the reporter molecule, wherein the lipophilic groups, when present in a live cell, are cleaved resulting in two or more negatively charged groups.

FIELD OF THE INVENTION

The present invention provides intracellular ion indicator compoundscapable of chelating and detecting metal ions in cells. The compoundsgenerally comprise a metal chelating moiety (M_(c)), a reporter moleculeand one or more lipophilic groups (G_(L)) covalently bonded to thereporter molecule, wherein the lipophilic groups, when present in a livecell, are cleaved resulting in one or more negatively charged groups.

BACKGROUND OF THE INVENTION

Metal ions such as calcium are involved in many cellular processesincluding signal transduction. Small variances in intracellular ionlevels can have a major impact on cellular processes. Measurement of ionlevels provides a very sensitive method for identifying various cellularactivities.

Several fluorescent calcium indicators known in art are employed inbiological research and high throughput screening. Generally, longwavelength indicators, such as rhodamine-based compounds bear a positivecharge. Positively charged molecules compartmentalize in cellmitochondria. Because calcium ion release in activated cells happens inthe cytosol, positively charged indicators show a weak response tocalcium ion influx. Alternatively, fluorescein-based indicators havealso been described that avoid accumulation in the mitochondria, yethave a shorter wavelength with less optimal emission spectra. Severalcalcium ion indicators are described in: Haugland, R. P. Handbook ofFluorescent Probes and Research Products, 9^(th) Ed, Molecular Probes:Eugene, Oreg., 2002, Chapter 20; Martin et al., Cell Calcium 2004, 36,509-14; and Beierlein et al., J. Neurophysiol. 2004, 92, 591-599.

Free cytosolic calcium ions play a key role in many aspects of cellularsignalling and regulation, and fluorogenic calcium indicators likeFluo-4 (Invitrogen Corp.) provide quantitative and spatial informationon calcium gradients with microscopic and plate reader measurements.Currently, green channel indicators like Fluo-4 enjoy a centerpiece rolein a variety of investigative methodologies and HTS assays at Gi, Go,and Gs coupled G Protein Coupled Receptors (GPCRs).

Shifting the excitation of the fluorescent calcium indicator towardlonger wavelengths would be beneficial for imaging applications bymaking it possible to multiplex the dye with existing green fluorophoresand fluorescent protein constructs. It could also improve readout in HTSassays by shifting to wavelengths where compound libraryautofluorescence is less of a problem.

A need exists for compounds which have the advantage of a longerwavelength and avoid localization in the mitochondria. Furthermore, aneed exists for fluorogenic probes that are taken up by cells andprovide sensitive detection of cytosolic metal ions, such as calcium,when in the cell. Here we report the development and evaluation of anovel fluorescent calcium indicators having better loading and responsecharacteristics than existing longer-wavelength calcium indicators.

SUMMARY OF THE INVENTION

The invention involves the use of dye molecules having a cleavablelipophilic group that masks negative charges on the dye. Upon cellularinternalization, the lipophilic groups are cleaved and the dye remainsat the site of action. Particularly, the negative charges decrease thedegree of mitochondrial localization.

The synthetic methods used in this invention are flexible and allowintroduction of a several extra acidic groups, which unlike thecarboxylates on certain dyes (such as BAPTA) serve the purpose ofpositive charge compensation, rather than participation in the chelateformation.

Also, because of extra negative charges, the compounds are less prone toleak into extracellular environment. The synthesis of low leakingindicators is a task of general importance. Therefore, the scope of thisinvention also includes carboxylic derivative of calcium indicatorshaving shorter-wavelength fluorophores.

Additionally, longwavelength indicators (generally having an emissionspectra above about 530 nm) described herein avoid cellularautofluorescence and interference from other agents such as drugs whichhave been introduced to the cell. The long wavelength intracellular ionindicators also allow for detection in multiplexing applications, suchas with GFP's or other indicators/dyes.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a spectroscopic study of the calcium binding to compound145 (λ_(ex)=555 nm, λ_(em)=580 nm; K_(d)=380 nmol), wherein FIG. 1Ashows changes in emission spectrum upon increase of [Ca²⁺] concentrationand FIG. 1B shows determination of the binding constant

FIG. 2 depicts Imaging of cellular localization of, wherein FIG. 2Ashows conventional red Ca indicator Rhod-2 (mitochondrial localization),FIG. 2B shows compound 146 (red cytosolic signal), and FIG. 2C showscytosolic indicator Fluo-4 (green cytosolic signal).

FIG. 3 shows detection of intracellular calcium after a (FIG. 3A) 1 uMfinal carbachol stimulation and (FIG. 3B) a dose-response study ofcarbachol in CHOM1 cells loaded as above with 10 and 20 uM Rhod-2 vs 10and 20 uM Compound 146.

DETAILED DESCRIPTION

Introduction

The present invention is based upon the phenomenon in which the opticalproperties of a fluorophore can be modulated by strategic covalentattachment of a metal ion-binding moiety (a chelator). Certain ionchelators reduce the fluorescence of the fluorophore by a through-spaceor through-bond interaction known as PET (photoinduced electrontransfer), in which fluorescence is inhibited by interaction of theexcited state fluorophore with an electron-rich chelator moiety. As thechelator moiety binds metal ion(s), the PET effect is diminished,resulting in increased fluorescence from the fluorophore.

This phenomenon has been employed in many commercially availableintracellular ion indicators, most notably in the detection ofintracellular calcium ions. However, these indicators each have theiradvantages and disadvantages. The green indicators are typically basedon a fluorescein dye and as such are well retained in the cytosol (dueto the overall negative charge) but have limited ability to bemultiplexed with other intracellular reporter molecules due to spectraloverlap. The longer wavelength intracellular ion indicators aretypically based on rhodamine, or other dyes comprising one or morepositive charges, that preferentially localize to specific organellessuch as the mitochondria, resulting in poor ion detection in thecytosol.

Herein we report on novel intracellular ion indicators that have a longwavelength emission spectra and are also well retained in the cytosol,making it possible for the first time to accurately measure cytosolicion concentration in a multiplexed assay such as with Green FluorescentProtein (GFP) or other well know green intracellular ion indicators. Ourinvention is based, in part, on the strategic placement of labilelipophilic groups on the dye portion of the intracellular ion indicator.While red ion indicators such as Rhod-2 comprise lipophilic groups theresulting negative charges on the chelating moiety of the compound isnot enough to retain the compound in the cytosol. We have found,unexpectedly, that by adding labile lipohilic groups, such as AM esters,to the fluorophore portion of the compound that these ion indicators areretained in the cytosol and not targeted to the mitochondria (FIG. 2).

The invention addresses the molecular design of fluorescentintracellular ion indicators intended for intracellular detection ofsuch ions. Introduction of extra negatively charged groups, such ascarboxylic acidic moieties into the indicator molecules resulted in anoverall signal increase due to cytosolic localization/retention. Beforecytosolic localization however, negative charges can inhibit or precludecellular internalization. Accordingly, addition of labile lipophilicgroups that are cleaved upon cellular internalization permittedtraversal of the plasma membrane. The compounds described herein aremore effective at localizing to the cytosol for ion detection thanexisting indicators and have an emission spectra longer than about 530nm to about 800 nm.

DEFINITIONS

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to specific compositionsor process steps, as such may vary. It must be noted that, as used inthis specification and the appended claims, the singular form “a”, “an”and “the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “a metal chelator” includes aplurality of chelators and reference to “a cell” includes a plurality ofcells and the like.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention is related. The following terms aredefined for purposes of the invention as described herein.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are encompassedwithin the scope of the present invention. Certain compounds of thepresent invention may exist in multiple crystalline or amorphous forms.In general, all physical forms are equivalent for the uses contemplatedby the present invention and are intended to be within the scope of thepresent invention.

Certain compounds of the present invention possess asymmetric carbonatoms (optical centers) or double bonds; the racemates, diastereomers,geometric isomers and individual isomers are encompassed within thescope of the present invention.

The compounds of the invention may be prepared as a single isomer (e.g.,enantiomer, cis-trans, positional, diastereomer) or as a mixture ofisomers. In a preferred embodiment, the compounds are prepared assubstantially a single isomer. Methods of preparing substantiallyisomerically pure compounds are known in the art. For example,enantiomerically enriched mixtures and pure enantiomeric compounds canbe prepared by using synthetic intermediates that are enantiomericallypure in combination with reactions that either leave the stereochemistryat a chiral center unchanged or result in its complete inversion.Alternatively, the final product or intermediates along the syntheticroute can be resolved into a single stereoisomer. Techniques forinverting or leaving unchanged a particular stereocenter, and those forresolving mixtures of stereoisomers are well known in the art and it iswell within the ability of one of skill in the art to choose anappropriate method for a particular situation. See, generally, Furnisset al. (eds.), VOGEL'S ENCYCLOPEDIA OF PRACTICAL ORGANIC CHEMISTRY5^(TH) ED., Longman Scientific and Technical Ltd., Essex, 1991, pp.809-816; and Heller, Acc. Chem. Res. 23: 128 (1990).

The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of the atoms thatconstitute such compounds. For example, the compounds may beradiolabeled with radioactive isotopes, such as for example tritium(³H), iodine-125 (¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations ofthe compounds of the present invention, whether radioactive or not, areintended to be encompassed within the scope of the present invention.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents, which would result from writing thestructure from right to left, e.g., —CH₂O— is intended to also recite—OCH₂—.

Any of the “substituted” groups may comprise a lipophilic group (G_(L)).

“Alkyl” refers to monovalent saturated aliphatic hydrocarbyl groupshaving from 1 to 10 carbon atoms and preferably 1 to 6 carbon atoms.This term includes, by way of example, linear and branched hydrocarbylgroups such as methyl (CH₃—), ethyl (CH₃CH₂—), n-propyl (CH₃CH₂CH₂—),isopropyl ((CH₃)₂CH—), n-butyl (CH₃CH₂CH₂CH₂—), isobutyl ((CH₃)₂CHCH₂—),sec-butyl ((CH₃)(CH₃CH₂)CH—), t-butyl ((CH₃)₃C—), n-pentyl(CH₃CH₂CH₂CH₂CH₂—), and neopentyl ((CH₃)₃CCH₂—).

“Substituted alkyl” refers to an alkyl group having from 1 to 5,preferably 1 to 3, or more preferably 1 to 2 substituents selected fromthe group consisting of alkoxy, substituted alkoxy, acyl, acylamino,acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl,aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy,aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, aryl,substituted aryl, aryloxy, substituted aryloxy, arylthio, substitutedarylthio, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxylester)oxy, cyano, cycloalkyl, substituted cycloalkyl, cycloalkyloxy,substituted cycloalkyloxy, cycloalkylthio, substituted cycloalkylthio,cycloalkenyl, substituted cycloalkenyl, cycloalkenyloxy, substitutedcycloalkenyloxy, cycloalkenylthio, substituted cycloalkenylthio,guanidino, substituted guanidino, halo, hydroxy, heteroaryl, substitutedheteroaryl, heteroaryloxy, substituted heteroaryloxy, heteroarylthio,substituted heteroarylthio, heterocyclic, substituted heterocyclic,heterocyclyloxy, substituted heterocyclyloxy, heterocyclylthio,substituted heterocyclylthio, nitro, SO₃H, substituted sulfonyl,sulfonyloxy, thioacyl, thiol, alkylthio, and substituted alkylthio,wherein said substituents are defined herein.

“Alkoxy” refers to the group —O-alkyl wherein alkyl is defined herein.Alkoxy includes, by way of example, methoxy, ethoxy, n-propoxy,isopropoxy, n-butoxy, t-butoxy, sec-butoxy, and n-pentoxy.

“Substituted alkoxy” refers to the group —O-(substituted alkyl) whereinsubstituted alkyl is defined herein.

“Acyl” refers to the groups H—C(O)—, alkyl-C(O)—, substitutedalkyl-C(O)—, alkenyl-C(O)—, substituted alkenyl-C(O)—, alkynyl-C(O)—,substituted alkynyl-C(O)—, cycloalkyl-C(O)—, substitutedcycloalkyl-C(O)—, cycloalkenyl-C(O)—, substituted cycloalkenyl-C(O)—,aryl-C(O)—, substituted aryl-C(O)—, heteroaryl-C(O)—, substitutedheteroaryl-C(O)—, heterocyclic-C(O)—, and substitutedheterocyclic-C(O)—, wherein alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic andsubstituted heterocyclic are as defined herein. Acyl includes the“acetyl” group CH₃C(O)—.

“Acylamino” refers to the groups —NRC(O)alkyl, —NRC(O)substituted alkyl,—NRC(O)cycloalkyl, —NRC(O)substituted cycloalkyl, —NRC(O)cycloalkenyl,—NRC(O)substituted cycloalkenyl, —NRC(O)alkenyl, —NRC(O)substitutedalkenyl, —NRC(O)alkynyl, —NRC(O)substituted alkynyl, —NRC(O)aryl,—NRC(O)substituted aryl, —NRC(O)heteroaryl, —NRC(O)substitutedheteroaryl, —NRC(O)heterocyclic, and —NRC(O)substituted heterocyclicwherein R is hydrogen or alkyl and wherein alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic andsubstituted heterocyclic are as defined herein.

“Acyloxy” refers to the groups alkyl-C(O)O—, substituted alkyl-C(O)O—,alkenyl-C(O)O—, substituted alkenyl-C(O)O—, alkynyl-C(O)O—, substitutedalkynyl-C(O)O—, aryl-C(O)O—, substituted aryl-C(O)O—, cycloalkyl-C(O)O—,substituted cycloalkyl-C(O)O—, cycloalkenyl-C(O)O—, substitutedcycloalkenyl-C(O)O—, heteroaryl-C(O)O—, substituted heteroaryl-C(O)O—,heterocyclic-C(O)O—, and substituted heterocyclic-C(O)O— wherein alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic are as definedherein.

“Amino” refers to the group —NH₂.

“Substituted amino” refers to the group —NR′R″ where R′ and R″ areindependently selected from the group consisting of hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, heteroaryl, substitutedheteroaryl, heterocyclic, substituted heterocyclic, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-alkenyl, —SO₂-substituted alkenyl,—SO₂-cycloalkyl, —SO₂-substituted cylcoalkyl, —SO₂-cycloalkenyl,—SO₂-substituted cycloalkenyl, —SO₂-aryl, —SO₂-substituted aryl,—SO₂-heteroaryl, —SO₂-substituted heteroaryl, —SO₂-heterocyclic, and—SO₂-substituted heterocyclic and wherein R′ and R″ are optionallyjoined, together with the nitrogen bound thereto to form a heterocyclicor substituted heterocyclic group, provided that R′ and R″ are both nothydrogen, and wherein alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, heterocyclic, and substitutedheterocyclic are as defined herein. When R′ is hydrogen and R″ is alkyl,the substituted amino group is sometimes referred to herein asalkylamino. When R′ and R″ are alkyl, the substituted amino group issometimes referred to herein as dialkylamino. When referring to amonosubstituted amino, it is meant that either R′ or R″ is hydrogen butnot both. When referring to a disubstituted amino, it is meant thatneither R′ nor R″ are hydrogen.

“Aminocarbonyl” refers to the group —C(O)NR′R″ where R′ and R″ areindependently selected from the group consisting of hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic and where R′ andR″ are optionally joined together with the nitrogen bound thereto toform a heterocyclic or substituted heterocyclic group, and whereinalkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic and substituted heterocyclic are asdefined herein.

“Aminothiocarbonyl” refers to the group —C(S)NR′R″ where R′ and R″ areindependently selected from the group consisting of hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic and where R′ andR″ are optionally joined together with the nitrogen bound thereto toform a heterocyclic or substituted heterocyclic group, and whereinalkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic and substituted heterocyclic are asdefined herein.

“Aminocarbonylamino” refers to the group —NRC(O)NR′R″ where R ishydrogen or alkyl and R′ and R″ are independently selected from thegroup consisting of hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedaryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic and where R′ and R″ are optionally joinedtogether with the nitrogen bound thereto to form a heterocyclic orsubstituted heterocyclic group, and wherein alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic andsubstituted heterocyclic are as defined herein.

“Aminothiocarbonylamino” refers to the group —NRC(S)NR′R″ where R ishydrogen or alkyl and R′ and R″ are independently selected from thegroup consisting of hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedaryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic and where R′ and R″ are optionally joinedtogether with the nitrogen bound thereto to form a heterocyclic orsubstituted heterocyclic group, and wherein alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic andsubstituted heterocyclic are as defined herein.

“Aminocarbonyloxy” refers to the group —O—C(O)NR′R″ where R′ and R″ areindependently selected from the group consisting of hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic and where R′ andR″ are optionally joined together with the nitrogen bound thereto toform a heterocyclic or substituted heterocyclic group, and whereinalkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic and substituted heterocyclic are asdefined herein.

“Aminosulfonyl” refers to the group —SO₂NR′R″ where R′ and R″ areindependently selected from the group consisting of hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic and where R′ andR″ are optionally joined together with the nitrogen bound thereto toform a heterocyclic or substituted heterocyclic group, and whereinalkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic and substituted heterocyclic are asdefined herein.

“Aminosulfonyloxy” refers to the group —O—SO₂NR′R″ where R′ and R″ areindependently selected from the group consisting of hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic and where R′ andR″ are optionally joined together with the nitrogen bound thereto toform a heterocyclic or substituted heterocyclic group, and whereinalkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic and substituted heterocyclic are asdefined herein.

“Aminosulfonylamino” refers to the group —NR—SO₂NR′R″ where R ishydrogen or alkyl and R¹⁰ and R¹¹ are independently selected from thegroup consisting of hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedaryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkyenyl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic and where R′ and R″ are optionally joinedtogether with the nitrogen bound thereto to form a heterocyclic orsubstituted heterocyclic group, and wherein alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkyenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic andsubstituted heterocyclic are as defined herein.

“Amidino” refers to the group —C(═Na′″)R′R″ where R′, R″, and R″ areindependently selected from the group consisting of hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic and where R′ andR″ are optionally joined together with the nitrogen bound thereto toform a heterocyclic or substituted heterocyclic group, and whereinalkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic and substituted heterocyclic are asdefined herein.

“Aryl” or “Ar” refers to a monovalent aromatic carbocyclic group of from6 to 14 carbon atoms having a single ring (e.g., phenyl) or multiplecondensed rings (e.g., naphthyl or anthryl) which condensed rings may ormay not be aromatic (e.g., 2-benzoxazolinone,2H-1,4-benzoxazin-3(4H)-one-7-yl, and the like) provided that the pointof attachment is at an aromatic carbon atom. Preferred aryl groupsinclude phenyl and naphthyl.

“Substituted aryl” refers to aryl groups which are substituted with 1 to5, preferably 1 to 3, or more preferably 1 to 2 substituents selectedfrom the group consisting of alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substitutedalkoxy, acyl, acylamino, acyloxy, amino, substituted amino,aminocarbonyl, aminothiocarbonyl, aminocarbonylamino,aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl,aminosulfonyloxy, aminosulfonylamino, amidino, aryl, substituted aryl,aryloxy, substituted aryloxy, arylthio, substituted arylthio, carboxyl,carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano,cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substitutedcycloalkyloxy, cycloalkylthio, substituted cycloalkylthio, cycloalkenyl,substituted cycloalkenyl, cycloalkenyloxy, substituted cycloalkenyloxy,cycloalkenylthio, substituted cycloalkenylthio, guanidino, substitutedguanidino, halo, hydroxy, heteroaryl, substituted heteroaryl,heteroaryloxy, substituted heteroaryloxy, heteroarylthio, substitutedheteroarylthio, heterocyclic, substituted heterocyclic, heterocyclyloxy,substituted heterocyclyloxy, heterocyclylthio, substitutedheterocyclylthio, nitro, SO₃H, substituted sulfonyl, sulfonyloxy,thioacyl, thiol, alkylthio, and substituted alkylthio, wherein saidsubstituents are defined herein.

“Aryloxy” refers to the group —O-aryl, where aryl is as defined herein,that includes, by way of example, phenoxy and naphthoxy.

“Substituted aryloxy” refers to the group —O-(substituted aryl) wheresubstituted aryl is as defined herein.

“Arylthio” refers to the group —S-aryl, where aryl is as defined herein.

“Substituted arylthio” refers to the group —S-(substituted aryl), wheresubstituted aryl is as defined herein.

“Alkenyl” refers to alkenyl groups having from 2 to 6 carbon atoms andpreferably 2 to 4 carbon atoms and having at least 1 and preferably from1 to 2 sites of alkenyl unsaturation. Such groups are exemplified, forexample, by vinyl, allyl, and but-3-en-1-yl.

“Substituted alkenyl” refers to alkenyl groups having from 1 to 3substituents, and preferably 1 to 2 substituents, selected from thegroup consisting of alkoxy, substituted alkoxy, acyl, acylamino,acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl,aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy,aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, aryl,substituted aryl, aryloxy, substituted aryloxy, arylthio, substitutedarylthio, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxylester)oxy, cyano, cycloalkyl, substituted cycloalkyl, cycloalkyloxy,substituted cycloalkyloxy, cycloalkylthio, substituted cycloalkylthio,cycloalkenyl, substituted cycloalkenyl, cycloalkenyloxy, substitutedcycloalkenyloxy, cycloalkenylthio, substituted cycloalkenylthio,guanidino, substituted guanidino, halo, hydroxy, heteroaryl, substitutedheteroaryl, heteroaryloxy, substituted heteroaryloxy, heteroarylthio,substituted heteroarylthio, heterocyclic, substituted heterocyclic,heterocyclyloxy, substituted heterocyclyloxy, heterocyclylthio,substituted heterocyclylthio, nitro, SO₃H, substituted sulfonyl,sulfonyloxy, thioacyl, thiol, alkylthio, and substituted alkylthio,wherein said substituents are defined herein and with the proviso thatany hydroxy substitution is not attached to a vinyl (unsaturated) carbonatom.

“Alkynyl” refers to alkynyl groups having from 2 to 6 carbon atoms andpreferably 2 to 3 carbon atoms and having at least 1 and preferably from1 to 2 sites of alkynyl unsaturation.

“Substituted alkynyl” refers to alkynyl groups having from 1 to 3substituents, and preferably 1 to 2 substituents, selected from thegroup consisting of alkoxy, substituted alkoxy, acyl, acylamino,acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl,aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy,aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, aryl,substituted aryl, aryloxy, substituted aryloxy, arylthio, substitutedarylthio, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxylester)oxy, cyano, cycloalkyl, substituted cycloalkyl, cycloalkyloxy,substituted cycloalkyloxy, cycloalkylthio, substituted cycloalkylthio,cycloalkenyl, substituted cycloalkenyl, cycloalkenyloxy, substitutedcycloalkenyloxy, cycloalkenylthio, substituted cycloalkenylthio,guanidino, substituted guanidino, halo, hydroxy, heteroaryl, substitutedheteroaryl, heteroaryloxy, substituted heteroaryloxy, heteroarylthio,substituted heteroarylthio, heterocyclic, substituted heterocyclic,heterocyclyloxy, substituted heterocyclyloxy, heterocyclylthio,substituted heterocyclylthio, nitro, SO₃H, substituted sulfonyl,sulfonyloxy, thioacyl, thiol, alkylthio, and substituted alkylthio,wherein said substituents are defined herein and with the proviso thatany hydroxy substitution is not attached to an acetylenic carbon atom.

“Carbonyl” refers to the divalent group —C(O)— which is equivalent to—C(═O)—.

“Carboxyl” or “carboxy” refers to —COOH or salts thereof.

“Carboxyl ester” or “carboxy ester” refers to the groups —C(O)O-alkyl,—C(O)O-substituted alkyl, —C(O)O-alkenyl, —C(O)O-substituted alkenyl,—C(O)O-alkynyl, —C(O)O-substituted alkynyl, —C(O)O-aryl,—C(O)O-substituted aryl, —C(O)O-cycloalkyl, —C(O)O-substitutedcycloalkyl, —C(O)O-cycloalkenyl, —C(O)O-substituted cycloalkenyl,—C(O)O-heteroaryl, —C(O)O-substituted heteroaryl, —C(O)O-heterocyclic,and —C(O)O-substituted heterocyclic wherein alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic are as defined herein.

“(Carboxyl ester)amino” refers to the group —NR—C(O)O-alkyl, substituted—NR—C(O)O-alkyl, —NR—C(O)O-alkenyl, —NR—C(O)O-substituted alkenyl,—NR—C(O)O-alkynyl, —NR—C(O)O-substituted alkynyl, —NR—C(O)O-aryl,—NR—C(O)O-substituted aryl, —NR—C(O)O-cycloalkyl, —NR—C(O)O-substitutedcycloalkyl, —NR—C(O)O-cycloalkenyl, —NR—C(O)O-substituted cycloalkenyl,—NR—C(O)O-heteroaryl, —NR—C(O)O-substituted heteroaryl,—NR—C(O)O-heterocyclic, and —NR—C(O)O-substituted heterocyclic wherein Ris alkyl or hydrogen, and wherein alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic are as defined herein.

“(Carboxyl ester)oxy” refers to the group —O—C(O)O-alkyl, substituted—O—C(O)O-alkyl, —O—C(O)O-alkenyl, —O—C(O)O-substituted alkenyl,—O—C(O)O-alkynyl, —O—C(O)O-substituted alkynyl, —O—C(O)O-aryl,—O—C(O)O-substituted aryl, —O—C(O)O-cycloalkyl, —O—C(O)O-substitutedcycloalkyl, —O—C(O)O-cycloalkenyl, —O—C(O)O-substituted cycloalkenyl,—O—C(O)O-heteroaryl, —O—C(O)O-substituted heteroaryl,—O—C(O)O-heterocyclic, and —O—C(O)O-substituted heterocyclic whereinalkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic, and substituted heterocyclic areas defined herein.

“Cyano” refers to the group —CN.

“Cycloalkyl” refers to cyclic alkyl groups of from 3 to 10 carbon atomshaving single or multiple cyclic rings including fused, bridged, andspiro ring systems. Examples of suitable cycloalkyl groups include, forinstance, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, andcyclooctyl.

“Cycloalkenyl” refers to non-aromatic cyclic alkyl groups of from 3 to10 carbon atoms having single or multiple cyclic rings and having atleast one >C═C< ring unsaturation and preferably from 1 to 2 sitesof >C═C< ring unsaturation.

“Substituted cycloalkyl” and “substituted cycloalkenyl” refers to acycloalkyl or cycloalkenyl group having from 1 to 5 or preferably 1 to 3substituents selected from the group consisting of oxo, thione, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino,substituted amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino,aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl,aminosulfonyloxy, aminosulfonylamino, amidino, aryl, substituted aryl,aryloxy, substituted aryloxy, arylthio, substituted arylthio, carboxyl,carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano,cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substitutedcycloalkyloxy, cycloalkylthio, substituted cycloalkylthio, cycloalkenyl,substituted cycloalkenyl, cycloalkenyloxy, substituted cycloalkenyloxy,cycloalkenylthio, substituted cycloalkenylthio, guanidino, substitutedguanidino, halo, hydroxy, heteroaryl, substituted heteroaryl,heteroaryloxy, substituted heteroaryloxy, heteroarylthio, substitutedheteroarylthio, heterocyclic, substituted heterocyclic, heterocyclyloxy,substituted heterocyclyloxy, heterocyclylthio, substitutedheterocyclylthio, nitro, SO₃H, substituted sulfonyl, sulfonyloxy,thioacyl, thiol, alkylthio, and substituted alkylthio, wherein saidsubstituents are defined herein.

“Cycloalkyloxy” refers to —O-cycloalkyl.

“Substituted cycloalkyloxy refers to —O-(substituted cycloalkyl).

“Cycloalkylthio” refers to —S-cycloalkyl.

“Substituted cycloalkylthio” refers to —S-(substituted cycloalkyl).

“Cycloalkenyloxy” refers to —O-cycloalkenyl.

“Substituted cycloalkenyloxy refers to —O-(substituted cycloalkenyl).

“Cycloalkenylthio” refers to —S-cycloalkenyl.

“Substituted cycloalkenylthio” refers to —S-(substituted cycloalkenyl).

“Guanidino” refers to the group —NHC(═NH)NH₂.

“Substituted guanidino” refers to —NR¹³C(═NR¹³)N(R¹³)₂ where each R¹³ isindependently selected from the group consisting of hydrogen, alkyl,substituted alkyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic and two R¹³groups attached to a common guanidino nitrogen atom are optionallyjoined together with the nitrogen bound thereto to form a heterocyclicor substituted heterocyclic group, provided that at least one R¹³ is nothydrogen, and wherein said substituents are as defined herein.

“H” indicates hydrogen.

“Halo” or “halogen” refers to fluoro, chloro, bromo and iodo.

“Hydroxy” or “hydroxyl” refers to the group —OH.

“Heteroaryl” refers to an aromatic group of from 1 to 10 carbon atomsand 1 to 4 heteroatoms selected from the group consisting of oxygen,nitrogen and sulfur within the ring. Such heteroaryl groups can have asingle ring (e.g., pyridinyl or furyl) or multiple condensed rings(e.g., indolizinyl or benzothienyl) wherein the condensed rings may ormay not be aromatic and/or contain a heteroatom provided that the pointof attachment is through an atom of the aromatic heteroaryl group. Inone embodiment, the nitrogen and/or the sulfur ring atom(s) of theheteroaryl group are optionally oxidized to provide for the N-oxide(N→O), sulfinyl, or sulfonyl moieties. Preferred heteroaryls includepyridinyl, pyrrolyl, indolyl, thiophenyl, and furanyl.

“Substituted heteroaryl” refers to heteroaryl groups that aresubstituted with from 1 to 5, preferably 1 to 3, or more preferably 1 to2 substituents selected from the group consisting of the same group ofsubstituents defined for substituted aryl.

“Heteroaryloxy” refers to —O-heteroaryl.

“Substituted heteroaryloxy refers to the group —O-(substitutedheteroaryl).

“Heteroarylthio” refers to the group —S-heteroaryl.

“Substituted heteroarylthio” refers to the group —S-(substitutedheteroaryl).

“Heterocycle” or “heterocyclic” or “heterocycloalkyl” or “heterocyclyl”refers to a saturated or unsaturated group having a single ring ormultiple condensed rings, including fused bridged and spiro ringsystems, from 1 to 10 carbon atoms and from 1 to 4 hetero atoms selectedfrom the group consisting of nitrogen, sulfur or oxygen within the ringwherein, in fused ring systems, one or more the rings can be cycloalkyl,aryl or heteroaryl provided that the point of attachment is through thenon-aromatic ring. In one embodiment, the nitrogen and/or sulfur atom(s)of the heterocyclic group are optionally oxidized to provide for theN-oxide, sulfinyl, sulfonyl moieties.

“Substituted heterocyclic” or “substituted heterocycloalkyl” or“substituted heterocyclyl” refers to heterocyclyl groups that aresubstituted with from 1 to 5 or preferably 1 to 3 of the samesubstituents as defined for substituted cycloalkyl.

“Heterocyclyloxy” refers to the group —O-heterocycyl.

“Substituted heterocyclyloxy refers to the group —O-(substitutedheterocycyl).

“Heterocyclylthio” refers to the group —S-heterocycyl.

“Substituted heterocyclylthio” refers to the group —S-(substitutedheterocycyl).

Examples of heterocycle and heteroaryls include, but are not limited to,azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine,pyridazine, indolizine, isoindole, indole, dihydroindole, indazole,purine, quinolizine, isoquinoline, quinoline, phthalazine,naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine,carbazole, carboline, phenanthridine, acridine, phenanthroline,isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine,imidazolidine, imidazoline, piperidine, piperazine, indoline,phthalimide, 1,2,3,4-tetrahydroisoquinoline,4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene,benzo[b]thiophene, morpholinyl, thiomorpholinyl (also referred to asthiamorpholinyl), 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidine,and tetrahydrofuranyl.

“Hydrazinyl” refers to the group —NHNH₂— or ═NNH—.

“Substituted hydrazinyl” refers to a hydrazinyl group, wherein anon-hydrogen atom, such as an alkyl group, is appended to one or both ofthe hydrazinyl amine groups. An example of substituted hydrazinyl is—N(alkyl)-NH₂ or ═N⁺(alkyl)-NH₂.

“Nitro” refers to the group —NO₂.

“Oxo” refers to the atom (═O) or (—O⁻).

“Spirocyclyl” refers to divalent saturated cyclic group from 3 to 10carbon atoms having a cycloalkyl or heterocyclyl ring with a spiro union(the union formed by a single atom which is the only common member ofthe rings) as exemplified by the following structure:

“Sulfonyl” refers to the divalent group —S(O)₂—.

“Substituted sulfonyl” refers to the group —SO₂-alkyl, —SO₂-substitutedalkyl, —SO₂-alkenyl, —SO₂-substituted alkenyl, —SO₂-cycloalkyl,—SO₂-substituted cylcoalkyl, —SO₂-cycloalkenyl, —SO₂-substitutedcycloalkenyl, —SO₂-aryl, —SO₂-substituted aryl, —SO₂-heteroaryl,—SO₂-substituted heteroaryl, —SO₂-heterocyclic, —SO₂-substitutedheterocyclic, wherein alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, heterocyclic and substitutedheterocyclic are as defined herein. Substituted sulfonyl includes groupssuch as methyl-SO₂—, phenyl-SO₂—, and 4-methylphenyl-SO₂—.

“Sulfonyloxy” refers to the group —OSO₂-alkyl, —OSO₂-substituted alkyl,—OSO₂-alkenyl, —OSO₂-substituted alkenyl, —OSO₂-cycloalkyl,—OSO₂-substituted cylcoalkyl, —OSO₂-cycloalkenyl, —OSO₂-substitutedcycloalkenyl, —OSO₂-aryl, —OSO₂-substituted aryl, —OSO₂-heteroaryl,—OSO₂-substituted heteroaryl, —OSO₂-heterocyclic, —OSO₂-substitutedheterocyclic, wherein alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, heterocyclic and substitutedheterocyclic are as defined herein.

“Thioacyl” refers to the groups H—C(S)—, alkyl-C(S)—, substitutedalkyl-C(S)—, alkenyl-C(S)—, substituted alkenyl-C(S)—, alkynyl-C(S)—,substituted alkynyl-C(S)—, cycloalkyl-C(S)—, substitutedcycloalkyl-C(S)—, cycloalkenyl-C(S)—, substituted cycloalkenyl-C(S)—,aryl-C(S)—, substituted aryl-C(S)—, heteroaryl-C(S)—, substitutedheteroaryl-C(S)—, heterocyclic-C(S)—, and substitutedheterocyclic-C(S)—, wherein alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic andsubstituted heterocyclic are as defined herein.

“Thiol” refers to the group —SH.

“Thiocarbonyl” refers to the divalent group —C(S)— which is equivalentto —C(═S)—.

“Thione” refers to the atom (═S).

“Alkylthio” refers to the group —S-alkyl wherein alkyl is as definedherein.

“Substituted alkylthio” refers to the group —S-(substituted alkyl)wherein substituted alkyl is as defined herein.

A dashed line projecting from a substituent, such as:

indicates the point of attachment to the base molecule. For a fusedring, dashed lines indicate portions of the base molecule where thefused ring is attached, such as:

wherein the full molecule could have the structure:

“Stereoisomer” or “stereoisomers” refer to compounds that differ in thechirality of one or more stereocenters. Stereoisomers includeenantiomers and diastereomers.

“Tautomer” refers to alternate forms of a compound that differ in theposition of a proton, such as enol-keto and imine-enamine tautomers, orthe tautomeric forms of heteroaryl groups containing a ring atomattached to both a ring —NH— moiety and a ring ═N— moeity such aspyrazoles, imidazoles, benzimidazoles, triazoles, and tetrazoles.

“Calcium sensitive dye” refers to a compound whose fluorescentproperties are affected, preferably increased, by the presence andcoordination of calcium ions.

The term “ion sensitive dye” as used herein refers to a compound whosefluorescent properties are affected, preferably increased, by thepresence and coordination of particular ions.

The term “intracellular ion sensitive dye”, “fluorogenic ion indicator”,or “fluorogenic intracellular ion indicator” are used interchangeablyand as used herein refer to a present compound comprising a reportermolecule and chelator moiety wherein the reporter molecule is appendedwith one or more labile lipophilic groups. Preferrably the reportermolecule has an emission spectra longer than about 530 nm.

“Patient,” “subject” or “individual” refers to mammals and includeshumans and non-human mammals, such as monkeys, dogs, cats, horses, cows,pocket pets, pigs or rats.

“Salt” refers to acceptable salts of a compound, which salts are derivedfrom a variety of organic and inorganic counter ions well known in theart and include, by way of example only, sodium, potassium, calcium,magnesium, ammonium, and tetraalkylammonium; and when the moleculecontains a basic functionality, salts of organic or inorganic acids,such as hydrochloride, hydrobromide, tartrate, mesylate, acetate,maleate, and oxalate.

“Treating” or “treatment” of a disease in a patient refers to 1)preventing the disease from occurring in a patient that is predisposedor does not yet display symptoms of the disease; 2) inhibiting thedisease or arresting its development; or 3) ameliorating or causingregression of the disease.

The terms “protein” and “polypeptide” are used herein in a generic senseto include polymers of amino acid residues of any length. The term“peptide” is used herein to refer to polypeptides having less than 250amino acid residues, typically less than 100 amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidues are an artificial chemical analogue of a correspondingnaturally occurring amino acid, as well as to naturally occurring aminoacid polymers.

The term “reactive group” as used herein refers to a group that iscapable of reacting with another chemical group to form a covalent bond,i.e. is covalently reactive under suitable reaction conditions, andgenerally represents a point of attachment for another substance. Thereactive group is a moiety, such as carboxylic acid or succinimidylester, on the compounds of the present invention that is capable ofchemically reacting with a functional group on a different compound toform a covalent linkage. Reactive groups generally include nucleophiles,electrophiles and photoactivatable groups.

Exemplary reactive groups include, but not limited to, olefins,acetylenes, alcohols, phenols, ethers, oxides, halides, aldehydes,ketones, carboxylic acids, esters, amides, cyanates, isocyanates,thiocyanates, isothiocyanates, amines, hydrazines, hydrazones,hydrazides, diazo, diazonium, nitro, nitriles, mercaptans, sulfides,disulfides, sulfoxides, sulfones, sulfonic acids, sulfinic acids,acetals, ketals, anhydrides, sulfates, sulfenic acids isonitriles,amidines, imides, imidates, nitrones, hydroxylamines, oximes, hydroxamicacids thiohydroxamic acids, allenes, ortho esters, sulfites, enamines,ynamines, ureas, pseudoureas, semicarbazides, carbodiimides, carbamates,imines, azides, azo compounds, azoxy compounds, and nitroso compounds.Reactive functional groups also include those used to preparebioconjugates, e.g., N-hydroxysuccinimide esters, maleimides and thelike. Methods to prepare each of these functional groups are well knownin the art and their application to or modification for a particularpurpose is within the ability of one of skill in the art (see, forexample, Sandler and Karo, eds., Organic Functional Group Preparations,Academic Press, San Diego, 1989).

The term “dye” as used herein refers to a compound that emits light toproduce an observable detectable signal.

The term “carrier molecule” as used herein refers to a biological or anon-biological component that is covalently bonded to a compound of thepresent invention. Such components include, but are not limited to, anamino acid, a peptide, a protein, a polysaccharide, a nucleoside, anucleotide, an oligonucleotide, a nucleic acid, a hapten, a psoralen, adrug, a hormone, a lipid, a lipid assembly, a synthetic polymer, apolymeric microparticle, a biological cell, a virus and combinationsthereof.

The term “Linker” or “L”, as used herein, refers to a single covalentbond or a series of stable covalent bonds incorporating 1-20 nonhydrogenatoms selected from the group consisting of C, N, O, S and P thatcovalently attach the fluorogenic or fluorescent compounds to anothermoiety such as a chemically reactive group or a biological andnon-biological component. Exemplary linking members include a moietythat includes —C(O)NH—, —C(O)O—, —NH—, —S—, —O—, and the like. A“cleavable linker” is a linker that has one or more cleavable groupsthat may be broken by the result of a reaction or condition. The term“cleavable group” refers to a moiety that allows for release of aportion, e.g., a fluorogenic or fluorescent moiety, of a conjugate fromthe remainder of the conjugate by cleaving a bond linking the releasedmoiety to the remainder of the conjugate. Such cleavage is eitherchemical in nature, or enzymatically mediated. Exemplary enzymaticallycleavable groups include natural amino acids or peptide sequences thatend with a natural amino acid.

In addition to enzymatically cleavable groups, it is within the scope ofthe present invention to include one or more sites that are cleaved bythe action of an agent other than an enzyme. Exemplary non-enzymaticcleavage agents include, but are not limited to, acids, bases, light(e.g., nitrobenzyl derivatives, phenacyl groups, benzoin esters), andheat. Many cleaveable groups are known in the art. See, for example,Jung et al., Biochem. Biophys. Acta, 761: 152-162 (1983); Joshi et al.,J. Biol. Chem., 265: 14518-14525 (1990); Zarling et al., J. Immunol.,124: 913-920 (1980); Bouizar et al., Eur. J. Biochem., 155: 141-147(1986); Park et al., J. Biol. Chem., 261: 205-210 (1986); Browning etal., J. Immunol., 143: 1859-1867 (1989). Moreover a broad range ofcleavable, bifunctional (both homo- and hetero-bifunctional) spacer armsare commercially available.

An exemplary cleavable group, an ester, is cleavable group that may becleaved by a reagent, e.g. sodium hydroxide, resulting in acarboxylate-containing fragment and a hydroxyl-containing product.

The linker can be used to attach the compound to another component of aconjugate, such as a targeting moiety (e.g., antibody, ligand,non-covalent protein-binding group, etc.), an analyte, a biomolecule, adrug and the like.

Unless indicated otherwise, the nomenclature of substituents that arenot explicitly defined herein are arrived at by naming the terminalportion of the functionality followed by the adjacent functionalitytoward the point of attachment. For example, the substituent“arylalkyloxycarbonyl” refers to the group (aryl)-(alkyl)-O—C(O)—.

It is understood that in all substituted groups defined above, polymersarrived at by defining substituents with further substituents tothemselves (e.g., substituted aryl having a substituted aryl group as asubstituent which is itself substituted with a substituted aryl group,which is further substituted by a substituted aryl group etc.) are notintended for inclusion herein. In such cases, the maximum number of suchsubstitutions is three. For example, serial substitutions of substitutedaryl groups with two other substituted aryl groups are limited to-substituted aryl-(substituted aryl)-substituted aryl.

Similarly, it is understood that the above definitions are not intendedto include impermissible substitution patterns (e.g., methyl substitutedwith 5 fluoro groups). Such impermissible substitution patterns are wellknown to the skilled artisan.

The term “affinity” as used herein refers to the strength of the bindinginteraction of two molecules, such as a metal chelating compound and ametal ion or a positively charged moiety and a negatively chargedmoiety.

The term “aqueous solution” as used herein refers to a solution that ispredominantly water and retains the solution characteristics of water.Where the aqueous solution contains solvents in addition to water, wateris typically the predominant solvent.

The term “cell permeable” as used herein refers to compounds of thepresent invention that are able to cross the cell membrane of livecells. Lipophilic groups that are covalently attached to the presentcompounds, facilitate this permeability and live cell entry. Once insidethe cells, the lipophilic groups are hydrolyzed resulting in chargedmolecules that are well retained in living cells. Particularly usefullipophilic groups include acetoxymethyl (AM) ester and acetate esterswherein once inside the cells the groups are cleaved by nonspecificesterases resulting in charged molecules.

The term “complex” as used herein refers to the association of two ormore molecules, usually by non-covalent bonding.

The term “detectable response” as used herein refers to a change in oran occurrence of, a signal that is directly or indirectly detectableeither by observation or by instrumentation and the presence ormagnitude of which is a function of the presence of a target metal ionin the test sample. Typically, the detectable response is an opticalresponse resulting in a change in the wavelength distribution patternsor intensity of absorbance or fluorescence or a change in light scatter,fluorescence quantum yield, fluorescence lifetime, fluorescencepolarization, a shift in excitation or emission wavelength or acombination of the above parameters. The detectable change in a givenspectral property is generally an increase or a decrease. However,spectral changes that result in an enhancement of fluorescence intensityand/or a shift in the wavelength of fluorescence emission or excitationare also useful. The change in fluorescence on ion binding is usuallydue to conformational or electronic changes in the indicator that mayoccur in either the excited or ground state of the fluorophore, due tochanges in electron density at the ion binding site, due to quenching offluorescence by the bound target metal ion, or due to any combination ofthese or other effects. Alternatively, the detectable response is anoccurrence of a signal wherein the fluorophore is inherently fluorescentand does not produce a change in signal upon binding to a metal ion orbiological compound.

The term “fluorophore” as used herein refers to a composition that isinherently fluorescent or demonstrates a change in fluorescence uponbinding to a biological compound or metal ion, or metabolism by anenzyme, i.e., fluorogenic. Fluorophores may be substituted to alter thesolubility, spectral properties or physical properties of thefluorophore. Numerous fluorophores are known to those skilled in the artand include, but are not limited to coumarin, acridine, furan, dansyl,cyanine, pyrene, naphthalene, benzofurans, quinolines, quinazolinones,indoles, benzazoles, borapolyazaindacenes, oxazine and xanthenes, withthe latter including fluoresceins, rhodamines, rosamine and rhodols aswell as other fluorophores described in RICHARD P. HAUGLAND, MOLECULARPROBES HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS (9^(th)edition, including the CD-ROM, September 2002). The fluorophore moietymay be substituted by substituents that enhance solubility, live cellpermeability and alter spectra absorption and emission.

The term “kit” as used refers to a packaged set of related components,typically one or more compounds or compositions.

The term “lipophilic group” as used herein refers to substituents whichmodify the charge or polarity of functionalities, such as an amino,thiol, hydroxyl, sulfonate or carboxylate group, thereby rendering themmore hydrophobic. Preferred protecting groups for use in the presentinvention include groups which reduce or eliminate the charge on theparent compound rendering it more lipophilic, and are also capable ofbeing cleaved, preferably in vivo by enzymes, such as esterases. Onepreferred protecting group is an acetoxymethyl (AM) ester or an analogthereof. Additional groups groups include, but are not limited to,substituted methyl and ethyl ethers such as, but not limited tomethoxymethyl ether, methythiomethyl ether, benzyloxymethyl ether,t-butoxymethyl ether, 2-methoxyethoxymethyl ether, tetrahydropyranylethers, 1-ethoxyethyl ether, allyl ether, benzyl ether; esters such as,but not limited to, benzoylformate, formate, acetate, trichloroacetate,and trifluoracetate; and silyl ethers such as those obtained by reactionof a hydroxyl group with a reagent such as, but not limited to,t-butyldimethyl-chlorosilane, trimethylchlorosilane,triisopropylchlorosilane, triethylchlorosilane. Modification to aminegroups include, but are not limited to, AM esters, amides such as,formamide, acetamide, trifluoroacetamide, and benzamide; imides, such asphthalimide, and dithiosuccinimide; and others. Modified sulfhydrylgroups include, but are not limited to, thioethers such as S-benzylthioether, and S-4-picolyl thioether; substituted S-methyl derivativessuch as hemithio, dithio and aminothio acetals; and others.

The term “metal chelator” or “metal chelating compound” as used hereinrefers to a chemical compound that combines with a metal ion to form achelate ring structure.

The term “metal ion” or “target metal ion” as used herein refers to anymetal cation that is capable of being chelated by a metal chelatingcompound. Typically, these metal ions are physiological and ornutritional relevant metal ion such as Na⁺, K⁺, Zn²⁺, Mg²⁺, Fe²⁺, andCa²⁺. The term metal ion used herein also refers to the metal ions Ga³⁺,Tb³⁺, La³⁺, Pb²⁺, Hg²⁺, Cd²⁺, Cu²⁺, Ni²⁺, Co²⁺, Mn²⁺, Ba²⁺, and Sr²⁺.Preferably the metal ion includes those that are present in the cytosolof a biological cell such as calcium, sodium, potassium, magnesium, zincand iron. Also included are metal ions that can act as a surrogate for aphysiological important metal ion, such as thallium, rubidium or lithiumwhich can act as a surrogate for potassium

The term “PET linker” refers to a linker as defined above, which affectsphotoinduced electron transfer (PET) between the chelator and dyemoieties. The PET linker is preferably a covalent bond. In anotherembodiment the PET linker is an alkylene spacer between the chelatingmoiety and the reporter moiety, thus limiting the interaction betweenthe ion sensor and reporter to the PET mechanism. Such linkers aredescribed more thoroughly in US Patent Publication No. 2006-0024833,U.S. Pat. Nos. 6,124,135; 6,359,135; He et al. Chem. Soc. (2003)125:1468-1469; and He et al. Anal. Chem. (2003) 75:3549-55, which areincorporated by reference (with respect to their disclosure of linkers,dyes and/or metal ion chelators) as if set forth fully herein.

The term “photoinduced electron transfer (PET)” as used herein refers tointramolecular electron transfer.

The term “reporter molecule” as used herein refers to a fluorophore ordye, terms that are defined above, which comprise part of the presentintracellular ion indicators.

The term “sample” as used herein refers to any material that may containtarget metal ions, as defined above. Typically, the sample is a livecell or a biological fluid that comprises endogenous host cell proteins.Alternatively, the sample may be a buffer solution or an environmentalsample containing target metal ions. The sample may be in an aqueoussolution, a viable cell culture or immobilized on a solid or semi solidsurface such as a polyacrylamide gel, membrane blot or on a microarray.

The term “Stokes shift” as used herein refers to the difference inwavelength between absorbed and emitted energy. Specifically, the Stokesshift is the difference (usually in frequency units) between thespectral positions and the band maxima (or band origin) of theabsorption and luminescence arising from the same electronictransitions.

Unless numbered, or proper claim construction (e.g. antecedent basis)requires it, multiple steps in a method or process claim/embodiment arenot required to be performed sequentially.

The Compounds

In general, for ease of understanding the present invention, the metalion binding compounds and corresponding substituents will first bedescribed in detail, followed by the many and varied methods in whichthe compounds find uses, which is followed by exemplified methods of useand synthesis of certain novel compounds that are particularlyadvantageous for use with the methods of the present invention.

The present compounds find utility in binding intracellular ions, inparticular calcium ions in a sample. The sample includes live cells or abiological fluid that comprises live cells and endogenous host cellproteins, buffer solutions and environmental samples. The sample mayalso comprise synthetic or artificial membranes or lipid bilayers.Therefore, the present compounds, when comprising a reporter molecule(typically a fluorophore or dye) find utility in binding, isolating,quantitating, monitoring, sequestering and detecting intracellular metalions wherein the detectable signal is modulated by photoinduced electrontransfer (PET). As described herein detection of the intracellular metalions is accomplished in live cells wherein the present compoundcomprises one or more labile lipophilic groups, such as an AM or acetateester that allows for entry across the live cell membrane, on thereporter molecule of the present compounds. Once inside the cellsnonspecific esterases cleave the AM or acetate ester resulting in acharged molecule that is well retained in the cell. These presentcompounds are particularly useful for binding physiologically relevantlevels of cytosolic metal ions.

The present compounds consist of three functional elements, metal ionchelating moiety (M_(c)), the reporter molecule (fluorescent dye orother moiety listed herein) and one or more lipophilic groups covalentlybonded to the reporter molecule through a linker wherein the lipophilicgroups, when present in a live cell, are cleaved resulting in one ormore negatively charged groups. One distinguishing feature of thesecompounds is one or more lipophilic groups (G_(L)) located on thereporter/dye molecule. The groups provide a number of technicaladvantages, including increased internalization in the cell, decreased“leaking” out of the cell, decreased localization in the mitochondria ofthe cell, use of reporter molecule skeletons that are typically neutralor positively charged, long wavelength cytosolic ion indicators, abilityto multiplex with shorter wavelength intracellular indicators, andthereby increased sensitivity for detection of cellularactivities/processes.

In a more specific embodiment, the present compound binds and detectscalcium ions wherein a detectable response is a result of photoinducedelectron transfer (PET) and the compound comprises a metal chelatingmoiety and a fluorophore or dye that is covalently bonded to the metalchelating moiety by a PET linker, such as a covalent bond or—(CR₂)_(n)NR′— or —(CR₂)_(n)— wherein R and R′ are independentlyselected from the group consisting of hydrogen, alkyl, and substitutedalkyl and n is 1-10. When the linker is —(CR₂)_(n)— the terminal carbonatom of the linker must be directly and covalently bonded to a nitrogenatom of the fluorophore.

In some embodiments, the present compounds exhibit a Stokes shift thatis greater than about 20 nm, 50 nm, or greater than about 100 nm, orgreater than about 150 nm.

One preferred compound involves an intracellular ion indicator compound,wherein the compound comprises a metal chelating moiety (M_(c)), areporter molecule (DYE) and two or more lipophilic groups (G_(L))covalently bonded through a linker to the reporter molecule, wherein thelipophilic groups, when present in a live cell, are cleaved resulting intwo or more negatively charged groups. In another embodiment thereof,the metal chelating moiety (M_(c)) is covalently bonded through a PETlinker to the reporter molecule. In another embodiment, the reportermolecule is a fluorescent dye. In another embodiment, the lipophilicgroups comprise an ester. More particularly, the two or more negativelycharged groups are carboxylate groups. In another embodiment, thelipophilic groups are selected from the group consisting of C₁-C₆carboxyalkyl, —(CH₂)₁₋₆COOCR¹⁵ ₂OC(═O)(CH₂)₀₋₄CH₃, andalpha-acyloxyalkyl; wherein R¹⁵ is H, alkyl or substituted alkyl. Inanother embodiment, the lipophilic groups are —CH₂CH₂COOCH—₂OC(═O)CH₃.In another embodiment the fluorescent dye comprises at least three or atleast four lipophilic groups. In another embodiment the fluorescent dyecomprises 2, 3, 4, 5, or 6 lipophilic groups. In another embodiment, theDYE is selected from the group consisting of a xanthene,borapolyazaindacene, cyanine, benzofuran, quinazolinone, indole,benzazole, oxazine, and a coumarin. In a preferred embodiment the DYE isa long wavelength such as those with an emission spectra longer thanabout 530 nm to 800 nm.

In another embodiment, the intracellular ion indicator compound has thestructure:(G_(L))_(v)-L-DYE-M_(c)

wherein,

L is a linker;

G_(L) is a lipophilic group;

DYE is a reporter molecule;

M_(c) is a metal chelating moiety; and

v is 2, 3, 4, or 5; or

v is 1, and DYE is a rhodamine a 3,6-diaminoxanthene (rhodamine).

In another embodiment, the intracellular ion indicator compound has thestructure:

-   wherein h and i are independently 0-4;-   z is 0 or 1; and-   AM is —CH₂OC(═O)CH₃.

In another embodiment of the invention, the intracellular ion indicatorfurther comprises a reactive group, carrier molecule or solid support.These substituents can be attached to the reporter molecule, the metalchelating moiety, provided they do not interfere with the coordinationof the metal ions, or to the linker.

Chelating Moiety

The ion-sensing or chelating moiety of the present compound is a moietythat will bind or chelate metal ions. Typically this results in a changein the fluorescent signal. Metal ions of the present invention, includebut are not limited to, Ca²⁺, Zn²⁺, Mg²⁺, Ga³⁺, Tb³⁺, La³⁺, Pb²⁺, Hg²⁺,Cd²⁺, Cu²⁺, Ni²⁺, Co²⁺, Fe²⁺, Mn²⁺, Ba²⁺, and Sr²⁺. In one aspect themetal ion is a physiological relevant ion selected from the groupconsisting of Ca²⁺, Mg²⁺, Fe²⁺ and Zn²⁺. In a further aspect the metalion is Ca²⁺, which is most notably chelated by the well-known BAPTAchelating moiety. In other embodiments, the metal ion is chelated by acrown ether, cryptan, APTRA (aminophenol triacetic acid) especially forcoordinating Mg²⁺, FluoZin-1, 2 or 3 or a phenanthroline, for example:

which may be further substituted.

The term “BAPTA” as used herein refers to a metal-chelating compoundthat is 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid or itsanalogs, derivatives, ring-fused variants and conjugates, and allmetallic and nonmetallic salts, partial salts and hydrates thereof,including any corresponding compounds disclosed in U.S. Pat. Nos.4,603,209; 4,849,362; 5,049,673; 5,453,517; 5,459,276; 5,516,911;5,501,980; 6,162,931 and 5,773,227. When used generically, “BAPTA”refers to two benzene rings that are joined by a C₁-C₃ hydrocarbonbridge terminated by oxygen atoms, including methylenedioxy (—OCH₂O—),ethylenedioxy (—OCH₂CH₂O—) or propylenedioxy (—OCH₂CH₂CH₂O) bridginggroups, where each benzene ring is optionally substituted by one or moresubstituents that adjust the metal ion-binding affinity, solubility,chemical reactivity, spectral properties or other physical properties ofthe compound. BAPTA derivatives additionally include compounds in whichthe benzene rings of the BAPTA structure are substituted by or fused toadditional aromatic, or heteroaromatic rings. In one embodiment thechelating group or BAPTA is substituted with a reactive group, carriermolecule, or solid support.

In one embodiment, the chelating moiety (M_(c)) is capable of bindingintracellular metal ion. In another embodiment, M_(c) comprises BAPTA ora crown ether moiety. More particularly, M_(c) has the structure:

wherein,

R¹, R², R³ and R⁴ are each independently selected from the groupconsisting of carbonyl, substituted carbonyl, carboxyl, alkyl,substituted alkyl, reactive group, carrier molecule, solid support, and-L-(G_(L))_(w); or

R² and R⁴ are bound together by: —(CH₂CH₂—O)_(t)—CH₂CH₂—, wherein t is 1or 2;

R⁵ and R⁶ are each independently selected from the group consisting of-L-(G_(L))_(w), alkyl, substituted alkyl, carbonyl, substitutedcarbonyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino,substituted amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino,aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl,aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester,(carboxyl ester)amino, (carboxyl ester)oxy, cyano, halo, hydroxy, nitro,SO₃ ⁻, sulfonyl, substituted sulfonyl, sulfonyloxy, thioacyl, thiol,alkylthio, substituted alkylthio, aryl, substituted aryl, heteroaryl,substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, reactive group, carrier molecule, solid support, andsubstituted heterocyclyl;

L is a linker;

G_(L) is a lipophilic group;

w is 1 or 2; and

m and n are each independently 0, 1, 2, or 3.

In a more particular embodiment thereof, n is 1 and R⁵ is —CH₃. Inanother embodiment, R² and R⁴ are bound together by: —CH₂CH₂—O—CH₂CH₂—.In another embodiment, R¹, R², R³ and R⁴ are -L-(G_(L))_(w). In anotherembodiment, L is a single covalent bond, or a covalent linkage that islinear or branched, cyclic or heterocyclic, saturated or unsaturated,having 1-30 nonhydrogen atoms selected from the group consisting of C,N, P, O and S; and are composed of any combination of ether, thioether,amine, ester, carboxamide, sulfonamide, hydrazide bonds and aromatic orheteroaromatic bonds. In another embodiment, L is -oxo-, alkoxy,-amino-, or -substituted amino-.

In another embodiment, M_(c) has the structure:

wherein,

X is —(CH₂CH₂—O)_(y)—CH₂CH₂—, wherein y is 1, 2, 3, 4, or 5;

R¹ is H, alkyl, substituted alkyl, carbonyl, reactive group, carriermolecule, solid support, or substituted carbonyl;

R⁶ is selected from the group consisting of -L-(G_(L))_(w), alkyl,substituted alkyl, carbonyl, substituted carbonyl, alkoxy, substitutedalkoxy, acyl, acylamino, acyloxy, amino, substituted amino,aminocarbonyl, aminothiocarbonyl, aminocarbonylamino,aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl,aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester,(carboxyl ester)amino, (carboxyl ester)oxy, cyano, halo, hydroxy, nitro,SO₃ ⁻, sulfonyl, substituted sulfonyl, sulfonyloxy, thioacyl, thiol,alkylthio, substituted alkylthio, aryl, substituted aryl, heteroaryl,substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, reactive group, carrier molecule, solid support, andsubstituted heterocyclyl;

L is a linker;

w is 1 or 2;

G_(L) is a lipophilic group; and

m is 0, 1, 2, or 3.

The present invention also provides zinc-binding compounds and methodsfor the selective binding and detection of physiological concentrationsof zinc ions. The zinc-binding compounds find utility in binding(sequestering), detecting (monitoring and/or quantitating) free zincions. Detection also includes the screening of drug candidates thataffect intracellular zinc ion concentrations, ion channels andzinc-binding proteins).

The metal chelating moiety of the zinc-binding compound is an analog ofthe well-known calcium chelator, BAPTA(1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid), wherein thechelating moiety has been modified from a tetraacetic acid moiety to atriacetic, diacetic or monoacetic acid moiety. This change in aceticacid groups on the metal chelating moiety results in the selectivebinding of zinc ions in the presence of calcium ions, both of which arepresent in biological fluids and intracellular cytosolic fluid andorganelles. Due to the relatively high concentration of physiologicalintracellular calcium compared to zinc and the fact that BAPTA bindscalcium with higher affinity than zinc, BAPTA is an ineffective chelatorfor binding physiological zinc ions in the presence of calcium ions.However, we found that by lowering the affinity for calcium thattriacetic acid analog of BAPTA chelating moieties preferentially bindzinc in the presence of calcium ions at physiological concentrations ofboth ions.

Accordingly, one particular embodiment of the present invention involvesdetection or monitoring for both zinc and calcium ions in the cytosol ofa cell, wherein at least two different intracellular ion indicators areintroduced to the cell and wherein each indicator comprises a dye whichis separately detectable from the other.

Reporter Molecule

The reporter molecule of the present invention functions as a reportermolecule to confer a detectable signal, directly or indirectly, to theintracellular metal ions. This results in the ability to detect, monitorand quantitate target metal ions in a sample.

The present reporter molecules can be any reporter molecule known to oneskilled in the art. A wide variety of chemically reactive fluorescentdyes that may be suitable for incorporation into the compounds of theinvention are already known in the art (RICHARD P. HAUGLAND, MOLECULARPROBES HANDBOOK OF FLUORESCENT PROBES AND RESEARCH PRODUCTS (2002)).Reporter molecules include, without limitation, a fluorophore, a dye, ora tandem dye (energy transfer pair). Preferably, the reporter moleculeis a fluorophore wherein when the present compounds are non-fluorescentuntil bound by a metal ion, i.e. fluorogenic. After binding a metal ionand upon illumination with an appropriate wavelength the compoundproduces a detectable signal modulated by PET.

Where the detectable response is a fluorescence response, it istypically a change in fluorescence, such as a change in the intensity,excitation or emission wavelength, distribution of fluorescence,fluorescence lifetime, fluorescence polarization, or a combinationthereof. Preferably, the detectable optical response upon binding atarget ion is a change in fluorescence intensity that is greater thanapproximately 150% relative to the same compound in the absence of themetal ion, more preferably greater than 5-fold, and most preferably morethat 10-fold.

A fluorescent dye of the present invention is any chemical moiety thatexhibits an absorption maximum beyond 280 nm, and when covalently linkedto a metal chelating moiety of the present invention, forms a presentfluorogenic metal ion-binding compound with an emission spectra longerthan about 530 nm. A preferred embodiment for detecting intracellularions in live cells is a fluorogenic ion-binding compound wherein thereporter molecule is fluorophore wherein the fluorophore is appendedwith one or more lipophilic groups. Most preferably are fluorescent dyesthat emit beyond the green portion of the spectrum (above 530 nm).

Dyes of the present invention include those derivatives that emit longerthan about 530 nm. These dyes and dye derivatives include, withoutlimitation, a pyrene, an anthracene, a naphthalene, an acridine, astilbene, an indole or benzindole, an oxazole or benzoxazole, a thiazoleor benzothiazole, a 4-amino-7-nitrobenz-2-oxa-1,3-diazole (NBD), acarbocyanine (including any corresponding compounds in U.S. Ser. Nos.09/557,275; 09/968,401 and 09/969,853 and U.S. Pat. Nos. 6,403,807;6,348,599; 5,486,616; 5,268,486; 5,569,587; 5,569,766; 5,627,027 and6,048,982), a carbostyryl, a porphyrin, a salicylate, an anthranilate,an azulene, a perylene, a pyridine, a quinoline, a borapolyazaindacene(including any corresponding compounds disclosed in U.S. Pat. Nos.4,774,339; 5,187,288; 5,248,782; 5,274,113; and 5,433,896), a xanthene(including any corresponding compounds disclosed in U.S. Pat. Nos.6,162,931; 6,130,101; 6,229,055; 6,339,392; 5,451,343 and U.S. Ser. No.09/922,333), an oxazine or a benzoxazine, a carbazine (including anycorresponding compounds disclosed in U.S. Pat. No. 4,810,636), aphenalenone, a coumarin (including an corresponding compounds disclosedin U.S. Pat. Nos. 5,696,157; 5,459,276; 5,501,980 and 5,830,912), abenzofuran (including an corresponding compounds disclosed in U.S. Pat.Nos. 4,603,209 and 4,849,362) and benzphenalenone (including anycorresponding compounds disclosed in U.S. Pat. No. 4,812,409) andderivatives thereof. As used herein, oxazines include resorufins(including any corresponding compounds disclosed in U.S. Pat. No.5,242,805), aminooxazinones, diaminooxazines, and theirbenzo-substituted analogs.

Where the dye is a xanthene, the dye is preferably a rhodamine or rhodol(in conjunction with a phenyl on the metal chelator (M_(c)) includingany corresponding compounds disclosed in U.S. Pat. Nos. 5,227,487 and5,442,045), a rosamine or a rhodamine (including any correspondingcompounds in U.S. Pat. Nos. 5,798,276; 5,846,737; 5,847,162; 6,017,712;6,025,505; 6,080,852; 6,716,979; 6,562,632). As used herein, fluoresceinincludes benzo- or dibenzofluoresceins, seminaphthofluoresceins, ornaphthofluoresceins. Similarly, as used herein rhodol includesseminaphthorhodafluors (including any corresponding compounds disclosedin U.S. Pat. No. 4,945,171).

Preferred dyes of the invention include dansyl, xanthene, cyanine,borapolyazaindacene, pyrene, naphthalene, coumarin, oxazine andderivatives thereof. Preferred xanthenes are aminoxanthene (rhodamine orrhodol) and derivatives thereof, BODIPY and dansyl.

Typically the dye contains one or more aromatic or heteroaromatic rings,that are optionally substituted one or more times by a variety ofsubstituents, including without limitation, halogen, nitro, sulfo,cyano, alkyl, perfluoroalkyl, alkoxy, alkenyl, alkynyl, cycloalkyl,arylalkyl, acyl, aryl or heteroaryl ring system, benzo, or othersubstituents typically present on chromophores or fluorophores known inthe art.

In an exemplary embodiment, the dyes are independently substituted bysubstituents selected from the group consisting of hydrogen, halogen,amino, substituted amino, alkyl, substituted alkyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, alkoxy, sulfo, reactive groupand carrier molecule. In another embodiment, the xanthene dyes of thisinvention comprise both compounds substituted and unsubstituted on thecarbon atom of the central ring of the xanthene by substituentstypically found in the xanthene-based dyes such as phenyl andsubstituted-phenyl moieties. Most preferred dyes are rhodamine, rhodol,and derivatives thereof. The choice of the dye attached to the chelatingmoiety will determine the metal ion-binding compound's absorption andfluorescence emission properties as well as its live cell properties,i.e. ability to localize to the cytosol.

In another preferred embodiment, the fluorescent dye comprises a3-aminoxanthene or a tautomer thereof. In another embodiment, thefluorescent dye comprises a 3,6-diaminoxanthene or a tautomer thereof.

In another embodiment, the fluorescent dye has the structure:

wherein,

R⁹ and R¹⁰ are each independently selected from the group consisting ofalkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino,acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl,aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy,aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl,carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, halo,hydroxy, nitro, SO₃ ⁻, sulfonyl, substituted sulfonyl, sulfonyloxy,thioacyl, thiol, alkylthio, substituted alkylthio, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, cycloalkyl, substitutedcycloalkyl, heterocyclyl, substituted heterocyclyl, reactive group,carrier molecule, solid support and -L-(G_(L))_(w);

R⁷ is ═O or ═N(R¹¹)₂, wherein R¹¹ is H, alkyl, (G_(L))_(w), substitutedalkyl, carbonyl, reactive group, carrier molecule, solid support orsubstituted carbonyl;

R⁸ is hydroxyl, —OR₁₃ or —N(R¹²)₂ wherein R¹² and R¹³ are independentlyH, alkyl, (G_(L))_(w), substituted alkyl, carbonyl, reactive group,carrier molecule, solid support or substituted carbonyl;

or one or both of R⁸ and (R¹⁰)_(p) and R⁷ and (R⁹)_(q) are takentogether to form a fused aryl or heteroaryl group;

L is a linker;

G_(L) is a lipophilic group;

w is 1 or 2; and

p and q are each independently 0, 1 or 2;

wherein the dye comprises at least two G_(L) groups.

In another embodiment, R⁷ is ═O, R⁸ is hydroxyl, p+q=1 and R⁹ or R¹⁰ is-L-(G_(L))_(w). In another embodiment, R⁹ is -L-(G_(L))_(w) and q is 1.

In another embodiment, L is a single covalent bond, or a covalentlinkage that is linear or branched, cyclic or heterocyclic, saturated orunsaturated, having 1-30 nonhydrogen atoms selected from the groupconsisting of C, N, P, O and S; and are composed of any combination ofether, thioether, amine, ester, carboxamide, sulfonamide, hydrazidebonds and aromatic or heteroaromatic bonds. In another embodiment, L isalkyl, substituted alkyl, -oxo-, alkoxy, -amino-, or -substitutedamino-.

In another embodiment, R⁷ is ═N⁺(G_(L))₂ and R⁸ is —OG_(L). In anotherembodiment, R⁷ is ═N⁺(G_(L))₂ and R⁸ is —N(G_(L))₂. More particularly,G_(L) is —(CH₂)₁₋₆COOCR¹⁵ ₂OC(═O)(CH₂)₀₋₄CH₃ wherein R¹⁵ is H, alkyl orsubstituted alkyl. More particular still, G_(L) is—CH₂CH₂COOCH₂OC(═O)CH₃.

In another embodiment, the fluorescent dye has the structure:

R⁹ and R¹⁰ are each independently selected from the group consisting ofalkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino,acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl,aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy,aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl,carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, halo,hydroxy, nitro, SO₃ ⁻, sulfonyl, substituted sulfonyl, sulfonyloxy,thioacyl, thiol, alkylthio, substituted alkylthio, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, cycloalkyl, substitutedcycloalkyl, heterocyclyl, substituted heterocyclyl and -L-(G_(L))_(w);

L is a linker;

G_(L) is a lipophilic group;

w is 1 or 2; and

p and q are each independently 0, 1 or 2.

In another embodiment thereof, G_(L) comprises an ester. Moreparticularly, G_(L) is selected from the group consisting of C₁-C₆carboxyalkyl, —(CH₂)₁₋₆COOCR¹⁵ ₂OC(═O)(CH₂)₀₋₄CH₃, andalpha-acyloxyalkyl; wherein R¹⁵ is H, alkyl or substituted alkyl. Moreparticularly, G_(L) is: —CH₂CH₂COOCH₂OC(═O)CH₃.

In another embodiment, the fluorescent dye is selected from the groupconsisting of a xanthene, borapolyazaindacene, cyanine, benzofuran,quinazolinone, indole, benzazole, oxazine, and a coumarin. Moreparticularly, the dye is a xanthene.

Linkers

The above described substituents are covalently attached to thechelating moiety or reporter moiety of the present compounds via alinker. In particular these substituents include a solid support,carrier molecule or reactive group wherein they may be directly attached(where Linker is a single bond) to the moieties (chelator or reporter)or attached through a series of stable bonds. When the linker is aseries of stable covalent bonds the linker typically incorporates 1-30nonhydrogen atoms selected from the group consisting of C, N, O, S andP. When the linker is not a single covalent bond, the linker may be anycombination of stable chemical bonds, optionally including, single,double, triple or aromatic carbon-carbon bonds, as well ascarbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds,sulfur-sulfur bonds, carbon-sulfur bonds, phosphorus-oxygen bonds,phosphorus-nitrogen bonds, and nitrogen-platinum bonds. Typically thelinker incorporates less than 15 nonhydrogen atoms and are composed ofany combination of ether, thioether, thiourea, amine, ester,carboxamide, sulfonamide, hydrazide bonds and aromatic or heteroaromaticbonds. Typically the linker is a combination of single carbon-carbonbonds and carboxamide, sulfonamide or thioether bonds. The bonds of thelinker typically result in the following moieties that can be found inthe linker: ether, thioether, carboxamide, thiourea, sulfonamide, urea,urethane, hydrazine, alkyl, aryl, heteroaryl, alkoky, cycloalkyl andamine moieties. Examples of a linker include substituted orunsubstituted polymethylene, arylene, alkylarylene, arylenealkyl, orarylthio.

In one embodiment, the linker contains 1-6 carbon atoms; in another, thelinker comprises a thioether linkage. Exemplary linking members includea moiety that includes —C(O)NH—, —C(O)O—, —NH—, —S—, —O—, and the like.In another embodiment, the linker is or incorporates the formula—(CH₂)_(d)(CONH(CH₂)_(e))_(z)— or where d is an integer from 0-5, e isan integer from 1-5 and z is 0 or 1. In a further embodiment, the linkeris or incorporates the formula —O—(CH₂)—. In yet another embodiment, thelinker is or incorporates a phenylene or a 2-carboxy-substitutedphenylene.

Any combination of linkers may be used to attach the carrier molecule,solid support or reactive group and the present compounds together. Thelinker may also be substituted to alter the physical properties of thereporter molecule or chelating moiety, such as spectral properties ofthe dye.

Another important feature of the linker is to provide an adequate spacebetween the carrier molecule, reactive group or solid support and thechelating moiety or reporter moiety so as to prevent steric hinderance.Therefore, the linker of the present compound is important for (1)attaching the carrier molecule, reactive group or solid support to thecompound, (2) providing an adequate space between the carrier molecule,reactive group or solid support and the compound so as not to stericallyhinder the action of the compound and (3) for altering the physicalproperties of the present compounds.

Reactive Groups

In another exemplary embodiment of the invention, the present compoundsare chemically reactive, and are substituted by at least one reactivegroup. The reactive group functions as the site of attachment foranother moiety, such as a carrier molecule or a solid support, whereinthe reactive group chemically reacts with an appropriate reactive orfunctional group on the carrier molecule or solid support. Thus, inanother aspect of the present invention the compounds comprise thechelating moiety, linker, reporter molecule, a reactive group moiety andoptionally a carrier molecule and/or a solid support.

In an exemplary embodiment, the compounds of the invention furthercomprise a reactive group which is a member selected from an acrylamide,an activated ester of a carboxylic acid, a carboxylic ester, an acylazide, an acyl nitrile, an aldehyde, an alkyl halide, an anhydride, ananiline, an amine, an aryl halide, an azide, an aziridine, a boronate, adiazoalkane, a haloacetamide, a haloalkyl, a halotriazine, a hydrazine,an imido ester, an isocyanate, an isothiocyanate, a maleimide, aphosphoramidite, a photoactivatable group, a reactive platinum complex,a silyl halide, a sulfonyl halide, and a thiol. In a particularembodiment the reactive group is selected from the group consisting ofcarboxylic acid, succinimidyl ester of a carboxylic acid, hydrazide,amine and a maleimide. In exemplary embodiment, at least one memberselected from R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹² and R¹³comprise a reactive group, carrier molecule or solid support.Preferably, at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ is areactive group, most preferred is at least one of R⁵, R⁶, R⁷ or W.Alternatively, if the present compound comprises a carrier molecule orsolid support a reactive group may be covalently attached independentlyto those substituents, allowing for further conjugation to a reportermolecule, carrier molecule or solid support.

In one aspect, the compound comprises at least one reactive group thatselectively reacts with an amine group. This amine-reactive group isselected from the group consisting of succinimidyl ester, sulfonylhalide, tetrafluorophenyl ester and iosothiocyanates. Thus, in oneaspect, the present compounds form a covalent bond with an aminecontaining molecule in a sample. In another aspect, the compoundcomprises at least one reactive group that selectively reacts with athiol group. This thiol-reactive group is selected from the groupconsisting of maleimide, haloalkyl and haloacetamide (including anyreactive groups disclosed in U.S. Pat. Nos. 5,362,628; 5,352,803 and5,573,904).

These reactive groups are synthesized during the formation of thepresent compound and carrier molecule and solid support containingcompounds to provide chemically reactive metal ion-binding compounds. Inthis way, compounds incorporating a reactive group can be covalentlyattached to a wide variety of carrier molecules or solid supports thatcontain or are modified to contain functional groups with suitablereactivity, resulting in chemical attachment of the components. In anexemplary embodiment, the reactive group of the compounds of theinvention and the functional group of the carrier molecule or solidsupport comprise electrophiles and nucleophiles that can generate acovalent linkage between them. Alternatively, the reactive groupcomprises a photoactivatable group, which becomes chemically reactiveonly after illumination with light of an appropriate wavelength.Typically, the conjugation reaction between the reactive group and thecarrier molecule or solid support results in one or more atoms of thereactive group being incorporated into a new linkage attaching thepresent compound of the invention to the carrier molecule or solidsupport. Selected examples of functional groups and linkages are shownin Table 1, where the reaction of an electrophilic group and anucleophilic group yields a covalent linkage.

TABLE 1 Examples of some routes to useful covalent linkagesElectrophilic Group Nucleophilic Group Resulting Covalent Linkageactivated esters* amines/anilines carboxamides acrylamides Thiolsthioethers acyl azides** amines/anilines carboxamides acyl halidesamines/anilines carboxamides acyl halides alcohols/phenols esters acylnitriles alcohols/phenols esters acyl nitriles amines/anilinescarboxamides aldehydes amines/anilines imines aldehydes or ketonesHydrazines hydrazones aldehydes or ketones hydroxylamines oximes alkylhalides amines/anilines alkyl amines alkyl halides Carboxylic acidsesters alkyl halides Thiols thioethers alkyl halides alcohols/phenolsethers alkyl sulfonates Thiols thioethers alkyl sulfonates Carboxylicacids esters alkyl sulfonates alcohols/phenols ethers anhydridesalcohols/phenols esters anhydrides amines/anilines carboxamides arylhalides Thiols thiophenols aryl halides Amines aryl amines aziridinesThiols thioethers boronates Glycols boronate esters carbodiimidesCarboxylic acids N-acylureas or anhydrides diazoalkanes Carboxylic acidsesters epoxides Thiols thioethers haloacetamides Thiols thioethershaloplatinate Amino platinum complex haloplatinate heterocycle platinumcomplex haloplatinate Thiol platinum complex halotriazinesamines/anilines aminotriazines halotriazines alcohols/phenols triazinylethers halotriazines Thiols triazinyl thioethers imido estersamines/anilines amidines isocyanates amines/anilines ureas isocyanatesalcohols/phenols urethanes isothiocyanates amines/anilines thioureasmaleimides Thiols thioethers phosphoramidites Alcohols phosphite esterssilyl halides Alcohols silyl ethers sulfonate esters amines/anilinesalkyl amines sulfonate esters Thiols thioethers sulfonate estersCarboxylic acids esters sulfonate esters Alcohols ethers sulfonylhalides amines/anilines sulfonamides sulfonyl halides phenols/alcoholssulfonate esters *Activated esters, as understood in the art, generallyhave the formula —COΩ, where Ω is a good leaving group (e.g.,succinimidyloxy (—OC₄H₄O₂) sulfosuccinimidyloxy (—OC₄H₃O₂—SO₃H),-1-oxybenzotriazolyl (—OC₆H₄N₃); or an aryloxy group or aryloxysubstituted one or more times by electron withdrawing substituents suchas nitro, fluoro, chloro, cyano, or trifluoromethyl, or combinationsthereof, used to form activated aryl esters; or a carboxylic acidactivated by a carbodiimide to form an anhydride or mixed anhydride—OCOR^(a) or —OCNR^(a)NHR^(b), where R^(a) and R^(b), which may be thesame or different, are C₁-C₆ alkyl, C₁-C₆ perfluoroalkyl, or C₁-C₆alkoxy; or cyclohexyl, 3-dimethylaminopropyl, or N-morpholinoethyl).**Acyl azides can also rearrange to isocyanates

Choice of the reactive group used to attach the compound of theinvention to the substance to be conjugated typically depends on thereactive or functional group on the substance to be conjugated and thetype or length of covalent linkage desired. The types of functionalgroups typically present on the organic or inorganic substances(biomolecule or non-biomolecule) include, but are not limited to,amines, amides, thiols, alcohols, phenols, aldehydes, ketones,phosphates, imidazoles, hydrazines, hydroxylamines, disubstitutedamines, halides, epoxides, silyl halides, carboxylate esters, sulfonateesters, purines, pyrimidines, carboxylic acids, olefinic bonds, or acombination of these groups. A single type of reactive site may beavailable on the substance (typical for polysaccharides or silica), or avariety of sites may occur (e.g., amines, thiols, alcohols, phenols), asis typical for proteins.

Typically, the reactive group will react with an amine, a thiol, analcohol, an aldehyde, a ketone, or with silica. Preferably, reactivegroups react with an amine or a thiol functional group, or with silica.In one embodiment, the reactive group is an acrylamide, an activatedester of a carboxylic acid, an acyl azide, an acyl nitrile, an aldehyde,an alkyl halide, a silyl halide, an anhydride, an aniline, an arylhalide, an azide, an aziridine, a boronate, a diazoalkane, ahaloacetamide, a halotriazine, a hydrazine (including hydrazides), animido ester, an isocyanate, an isothiocyanate, a maleimide, aphosphoramidite, a reactive platinum complex, a sulfonyl halide, or athiol group. By “reactive platinum complex” is particularly meantchemically reactive platinum complexes such as described in U.S. Pat.No. 5,714,327.

Where the reactive group is an activated ester of a carboxylic acid,such as a succinimidyl ester of a carboxylic acid, a sulfonyl halide, atetrafluorophenyl ester or an isothiocyanates, the resulting compound isparticularly useful for preparing conjugates of carrier molecules suchas proteins, nucleotides, oligonucleotides, or haptens. Where thereactive group is a maleimide, haloalkyl or haloacetamide (including anyreactive groups disclosed in U.S. Pat. Nos. 5,362,628; 5,352,803 and5,573,904 (supra)) the resulting compound is particularly useful forconjugation to thiol-containing substances. Where the reactive group isa hydrazide, the resulting compound is particularly useful forconjugation to periodate-oxidized carbohydrates and glycoproteins, andin addition is an aldehyde-fixable polar tracer for cell microinjection.Where the reactive group is a silyl halide, the resulting compound isparticularly useful for conjugation to silica surfaces, particularlywhere the silica surface is incorporated into a fiber optic probesubsequently used for remote ion detection or quantitation.

In a particular aspect, the reactive group is a photoactivatable groupsuch that the group is only converted to a reactive species afterillumination with an appropriate wavelength. An appropriate wavelengthis generally a UV wavelength that is less than 400 nm. This methodprovides for specific attachment to only the target molecules, either insolution or immobilized on a solid or semi-solid matrix.Photoactivatable reactive groups include, without limitation,benzophenones, aryl azides and diazirines.

Preferably, the reactive group is a photoactivatable group, succinimidylester of a carboxylic acid, a haloacetamide, haloalkyl, a hydrazine, anisothiocyanate, a maleimide group, an aliphatic amine, a silyl halide, acadaverine or a psoralen. More preferably, the reactive group is asuccinimidyl ester of a carboxylic acid, a maleimide, an iodoacetamide,or a silyl halide. In a particular embodiment the reactive group is asuccinimidyl ester of a carboxylic acid, a sulfonyl halide, atetrafluorophenyl ester, an iosothiocyanates or a maleimide.

Carrier Molecules

In any of the above embodiments, the compound can be covalently bound toa carrier molecule. If the compound has a reactive group, then thecarrier molecule can alternatively be linked to the compound through thereactive group. The reactive group may contain both a reactivefunctional moiety and a linker, or only the reactive functional moiety.

A variety of carrier molecules are useful in the present invention.Exemplary carrier molecules include antigens, steroids, vitamins, drugs,haptens, metabolites, toxins, environmental pollutants, amino acids,peptides, proteins, nucleic acids, nucleic acid polymers, carbohydrates,lipids, and polymers. In another exemplary embodiment, at least onemember selected from R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹²or R¹³ is a carrier molecule or is attached to a carrier molecule.

In an exemplary embodiment, the carrier molecule comprises an aminoacid, a peptide, a protein, a polysaccharide, a nucleoside, anucleotide, an oligonucleotide, a nucleic acid, a hapten, a psoralen, adrug, a hormone, a lipid, a lipid assembly, a synthetic polymer, apolymeric microparticle, a biological cell, a virus and combinationsthereof. In another exemplary embodiment, the carrier molecule isselected from a hapten, a nucleotide, an oligonucleotide, a nucleic acidpolymer, a protein, a peptide or a polysaccharide. In a preferredembodiment the carrier molecule is amino acid, a peptide, a protein, apolysaccharide, a nucleoside, a nucleotide, an oligonucleotide, anucleic acid, a hapten, a psoralen, a drug, a hormone, a lipid, a lipidassembly, a tyramine, a synthetic polymer, a polymeric microparticle, abiological cell, cellular components, an ion chelating moiety, anenzymatic substrate or a virus. In another preferred embodiment, thecarrier molecule is an antibody or fragment thereof, an antigen, anavidin or streptavidin, a biotin, a dextran, an IgG binding protein, afluorescent protein, agarose, and a non-biological microparticle.

In an exemplary embodiment, the enzymatic substrate is selected from anamino acid, peptide, sugar, alcohol, alkanoic acid, 4-guanidinobenzoicacid, nucleic acid, lipid, sulfate, phosphate, —CH₂OCOalkyl andcombinations thereof. Thus, the enzyme substrates can be cleave byenzymes selected from the group consisting of peptidase, phosphatase,glycosidase, dealkylase, esterase, guanidinobenzotase, sulfatase,lipase, peroxidase, histone deacetylase, endoglycoceramidase,exonuclease, reductase and endonuclease.

In another exemplary embodiment, the carrier molecule is an amino acid(including those that are protected or are substituted by phosphates,carbohydrates, or C₁ to C₂₂ carboxylic acids), or a polymer of aminoacids such as a peptide or protein. In a related embodiment, the carriermolecule contains at least five amino acids, more preferably 5 to 36amino acids. Exemplary peptides include, but are not limited to,neuropeptides, cytokines, toxins, protease substrates, and proteinkinase substrates. Other exemplary peptides may function as organellelocalization peptides, that is, peptides that serve to target theconjugated compound for localization within a particular cellularsubstructure by cellular transport mechanisms. Preferred protein carriermolecules include enzymes, antibodies, lectins, glycoproteins, histones,albumins, lipoproteins, avidin, streptavidin, protein A, protein G,phycobiliproteins and other fluorescent proteins, hormones, toxins andgrowth factors. Typically, the protein carrier molecule is an antibody,an antibody fragment, avidin, streptavidin, a toxin, a lectin, or agrowth factor. Exemplary haptens include biotin, digoxigenin andfluorophores.

In another exemplary embodiment, the carrier molecule comprises anucleic acid base, nucleoside, nucleotide or a nucleic acid polymer,optionally containing an additional linker or spacer for attachment of afluorophore or other ligand, such as an alkynyl linkage (U.S. Pat. No.5,047,519), an aminoallyl linkage (U.S. Pat. No. 4,711,955) or otherlinkage. In another exemplary embodiment, the nucleotide carriermolecule is a nucleoside or a deoxynucleoside or a dideoxynucleoside.

Exemplary nucleic acid polymer carrier molecules are single- ormulti-stranded, natural or synthetic DNA or RNA oligonucleotides, orDNA/RNA hybrids, or incorporating an unusual linker such as morpholinederivatized phosphates (AntiVirals, Inc., Corvallis Oreg.), or peptidenucleic acids such as N-(2-aminoethyl)glycine units, where the nucleicacid contains fewer than 50 nucleotides, more typically fewer than 25nucleotides.

In another exemplary embodiment, the carrier molecule comprises acarbohydrate or polyol that is typically a polysaccharide, such asdextran, FICOLL, heparin, glycogen, amylopectin, mannan, inulin, starch,agarose and cellulose, or is a polymer such as a poly(ethylene glycol).In a related embodiment, the polysaccharide carrier molecule includesdextran, agarose or FICOLL.

In another exemplary embodiment, the carrier molecule comprises a lipid(typically having 6-25 carbons), including glycolipids, phospholipids,and sphingolipids. Alternatively, the carrier molecule comprises a lipidvesicle, such as a liposome, or is a lipoprotein (see below). Somelipophilic substituents are useful for facilitating transport of theconjugated dye into cells or cellular organelles.

Alternatively, the carrier molecule is a cell, cellular systems,cellular fragment, or subcellular particles, including virus particles,bacterial particles, virus components, biological cells (such as animalcells, plant cells, bacteria, or yeast), or cellular components.Examples of cellular components that are useful as carrier moleculesinclude lysosomes, endosomes, cytoplasm, nuclei, histones, mitochondria,Golgi apparatus, endoplasmic reticulum and vacuoles.

In another exemplary embodiment, the carrier molecule non-covalentlyassociates with organic or inorganic materials. Exemplary embodiments ofthe carrier molecule that possess a lipophilic substituent can be usedto target lipid assemblies such as biological membranes or liposomes bynon-covalent incorporation of the dye compound within the membrane,e.g., for use as probes for membrane structure or for incorporation inliposomes, lipoproteins, films, plastics, lipophilic microspheres orsimilar materials.

In an exemplary embodiment, the carrier molecule comprises a specificbinding pair member wherein the present compounds are conjugated to aspecific binding pair member and used to the formation of the boundpair. Alternatively, the presence of the labeled specific binding pairmember indicates the location of the complementary member of thatspecific binding pair; each specific binding pair member having an areaon the surface or in a cavity which specifically binds to, and iscomplementary with, a particular spatial and polar organization of theother. In this instance, the dye compounds of the present inventionfunction as a reporter molecule for the specific binding pair. Exemplarybinding pairs are set forth in Table 2.

TABLE 2 Representative Specific Binding Pairs antigen Antibody biotinavidin (or streptavidin or anti-biotin) IgG* protein A or protein G drugdrug receptor folate folate binding protein toxin toxin receptorcarbohydrate lectin or carbohydrate receptor peptide peptide receptorprotein protein receptor enzyme substrate Enzyme DNA (RNA) cDNA (cRNA)†hormone hormone receptor ion Chelator *IgG is an immunoglobulin †cDNAand cRNA are the complementary strands used for hybridizationSolid Supports

In an exemplary embodiment, the present compounds of the invention arecovalently bonded to a solid support. The solid support may be attachedto the compound either through the chelating moiety, reporter moleculeor DYE, a substituent on the chelating moiety, reporter molecule or DYE,or through a reactive group, if present, or through a carrier molecule,if present. Even if a reactive group, reporter molecule and/or a carriermolecule are present, the solid support may be attached through thechelating moiety. In another exemplary embodiment, at least one memberselected from R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², and R¹³is a solid support or is attached to a solid support.

A solid support suitable for use in the present invention is typicallysubstantially insoluble in liquid phases. Solid supports of the currentinvention are not limited to a specific type of support. Rather, a largenumber of supports are available and are known to one of ordinary skillin the art. Thus, useful solid supports include solid and semi-solidmatrixes, such as aerogels and hydrogels, resins, beads, biochips(including thin film coated biochips), microfluidic chip, a siliconchip, multi-well plates (also referred to as microtitre plates ormicroplates), membranes, conducting and nonconducting metals, glass(including microscope slides) and magnetic supports. More specificexamples of useful solid supports include silica gels, polymericmembranes, particles, derivatized plastic films, glass beads, cotton,plastic beads, alumina gels, polysaccharides such as Sepharose,poly(acrylate), polystyrene, poly(acrylamide), polyol, agarose, agar,cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin,mannan, inulin, nitrocellulose, diazocellulose, polyvinylchloride,polypropylene, polyethylene (including poly(ethylene glycol)), nylon,latex bead, magnetic bead, paramagnetic bead, superparamagnetic bead,starch and the like.

In some embodiments, the solid support may include a solid supportreactive functional group, including, but not limited to, hydroxyl,carboxyl, amino, thiol, aldehyde, halogen, nitro, cyano, amido, urea,carbonate, carbamate, isocyanate, sulfone, sulfonate, sulfonamide,sulfoxide, etc., for attaching the compounds of the invention. Usefulreactive groups are disclosed above and are equally applicable to thesolid support reactive functional groups herein.

A suitable solid phase support can be selected on the basis of desiredend use and suitability for various synthetic protocols. For example,where amide bond formation is desirable to attach the compounds of theinvention to the solid support, resins generally useful in peptidesynthesis may be employed, such as polystyrene (e.g., PAM-resin obtainedfrom Bachem Inc., Peninsula Laboratories, etc.), POLYHIPE™ resin(obtained from Aminotech, Canada), polyamide resin (obtained fromPeninsula Laboratories), polystyrene resin grafted with polyethyleneglycol (TentaGel™, Rapp Polymere, Tubingen, Germany),polydimethyl-acrylamide resin (available from Milligen/Biosearch,California), or PEGA beads (obtained from Polymer Laboratories).

Preparation of Conjugates

The preparation of dye conjugates using reactive dyes or linkers is welldocumented, e.g. by R. Haugland, MOLECULAR PROBES HANDBOOK OFFLUORESCENT PROBES AND RESEARCH CHEMICALS, Chapters 1-3 (1996); andBrinkley, BIOCONJUGATE CHEM., 3, 2 (1992). Conjugates typically resultfrom mixing appropriate reactive dyes and the substance to be conjugatedin a suitable solvent in which both are soluble. The dyes of theinvention are readily soluble in aqueous solutions, facilitatingconjugation reactions with most biological materials. For those reactivedyes that are photoactivated, conjugation requires illumination of thereaction mixture to activate the reactive dye.

In-Situ Formation of Conjugates

In one embodiment, compounds and compositions comprising a reactivegroup passively diffuses into cells, wherein the reactive group reactswith intracellular amines, forming fluorescent conjugates that arewell-retained and can be fixed with aldehyde fixatives. Excessunconjugated reagent and by-products passively diffuse to theextracellular medium, where they can be washed away.

The indicator—protein adducts that form in labeled cells can be retainedby the cells throughout development, meiosis, and in vivo tracing. Thelabel is inherited by daughter cells after cell division, or cellfusion, and is not transferred to adjacent cells in a population.

Synthesis

A particular aspect of the invention provides a method of making aintracellular ion indicator compound described herein comprising:

contacting a metal ion chelator (M_(c)) with a reporter molecule, suchthat the reporter molecule covalently binds to the metal ion chelator;and

contacting a lipophilic group with a negatively charged substituentpresent on the reporter molecule, such that the lipophilic group bindscovalently to the negatively charged group thereby neutralizing thenegatively charged group.

In a more particular embodiment, the negatively charged group is acarboxylate. In another embodiment the reporter molecule is afluorescent dye. More particularly, the fluorescent dye is a xanthene,such as a 3-aminoxanthene or tatumter thereof.

In a more particular embodiment, the a metal ion chelator (M_(c))comprises a reactive group when it is contacted with the reportermolecule. More particular still, the reactive group is an aldehyde.

Additional synthetic methods and schemes contemplated as part of thepresent invention are provided in the Examples section.

Method of Use

The intracellular ion chelating compounds of the invention are usefulfor any application where it is desirable to complex a metal ion.Selected compounds of the invention may be useful as ionophores, thatis, they facilitate the transport of selected target ions across cellmembranes. Typically, however the present intracellular ion indicatorsare useful for detecting, monitoring, quantitating or sequestering ofintracellular metal ions that are found in the cytosol or surrogatemetal ions not normally found in the cytosol or other cellularcompartments. In a preferred embodiment the intracellular ion indicatorsare well retained in the cytosol or other discrete locations in he celland when coordinated with an appropriate metal ion emit at a wavelengthgreater than about 530 nm. Such intracellular metal ions includecalcium, magnesium, zinc and potassium as well as any metal ion that canact as a surrogate. Calcium is an important cytosolic metal ion and apreferred method is the use of the present compounds to detect thismetal ion using methods well known in the art.

In an alternative embodiment the present compounds are bound to acarrier molecule or solid support such as a bioparticle (e.g. bacterialparticles), peptides, antibodies, polymeric particles (e.g. polystyrenebeads), receptor binding domains, nucleic acid binding proteins, kinasesubstrates, phosphatase substrates, and other carrier molecules that areuseful for facilitating passive and cell mediated uptake of the presentcompounds or carrier molecules that are useful for localizing thepresent compounds such that measurement of the metal ions is indicativeof the cellular events in the local environment. Examples of carriermolecules that facilitate passive and cell mediated uptake includeantibodies, bioparticles, receptor binding proteins or peptides (bindingdomain) and the like. Examples of carrier molecules that provide usefulinformation either for localization or because they act as an enzymesubstrate include kinase substrates, phosphatase substrates, antibodies,nucleic acid binding proteins, nucleic acids and the like.

In order for a particular indicator of the present invention to beuseful for detection purposes, it must exhibit a detectable change inspectral properties upon coordination with an appropriate intracellularmetal ion. Preferably the change in spectral properties is a change influorescence properties. More preferably, the instant indicators displayan intensity increase or decrease in emission energy upon thecomplexation of the desired target ion.

The present compounds are useful for binding target ions resulting in acomplex of the target ion and the present compounds. Therefore, anadditional aspect of the invention includes the compound of theinvention further comprising a metal ion that is coordinated within thechelate portion of the compound. The metal ion is optionally Ca²⁺, Zn²⁺,Mg²⁺, Ga³⁺, Tb³⁺, La³⁺, Pb²⁺, Hg²⁺, Cd²⁺, Cu²⁺, Ni²⁺, Co²⁺, Fe²⁺, Mn²⁺,Ba²⁺, and Sr²⁺. Preferably the complex comprises physiological relevantcations such as Ca²⁺, Mg²⁺, Fe²⁺ and Zn²⁺. Most preferably, the complexcomprises calcium, magnesium, iron, zinc, sodium, potassium or thalliumions.

Accordingly, one aspect of the invention provides a method for bindingintracellular metal ions in a sample, comprising:

contacting the sample with a intracellular ion indicator compound,wherein the compound comprises a metal chelating moiety, a reportermolecule and one or more lipophilic groups covalently bonded to thereporter molecule through a linker wherein the lipophilic groups, whenpresent in a live cell, are enzymatically cleaved resulting in one ormore negatively charged groups; and

incubating the sample and compound for sufficient time to allow thecompound to

chelate the appropriate intracellular ion whereby the metal ion isbound.

In a more particular embodiment, the method further comprisesilluminating the compound with a suitable light source whereby theintracellular ion is detected. In this method the present compounds areincubated with the sample for a sufficient amount of time to allow thecompound to be either passively or by cell mediated mechanisms to betaken up by the biological cells. Once inside the cells the labilelipophile groups are enzymatically cleaved resulting in a compound thatis well retained in the cytosol or other discrete locations of the cell.The sample is then incubated for a sufficient amount of time to allowthe present compounds to chelate the appropriate intracellular metalion. In this instance, “appropriate metal ion” means an ion that isbound by the chelating moiety present on the intracellular ionindicator. This results in metal ions being bound in the cell.

Another aspect of the invention provides a method for detecting and/oridentifying intracellular metal ions in a sample, comprising:

contacting the sample with a intracellular ion indicator compound,wherein the compound comprises a metal chelating moiety, a reportermolecule and one or more lipophilic groups covalently bonded to thereporter molecule through a linker wherein the lipophilic groups, whenpresent in a live cell, are cleaved resulting in one or more negativelycharged groups;

incubating the sample for a period of time sufficient for the compoundto enter the cell;

cleaving the one or more lipophilic groups; and

illuminating the sample with an appropriate wavelength, wherein theintracellular metal ion is identified.

The sample is illuminated with an appropriate wavelength whereby thetarget ion is detected. In such an assay the target ion can also bequantitated and monitored.

The specific indicator used in an assay or experiment is selected basedon the desired affinity for the target ion as determined by the expectedconcentration range in the sample, the desired spectral properties, andthe desired selectivity. Initially, the suitability of a material as anindicator of ion concentration is commonly tested by mixing a constantamount of the indicating reagent with a measured amount of the targetion under the expected experimental conditions.

Preferred indicators display a high selectivity, that is, they show asufficient rejection of non-target ions. The interference of anon-target ion is tested by a comparable titration of the indicator withthat ion. Although preferred target ions for most indicators of thepresent invention are Ca⁺⁺ any ion that yields a detectable change inabsorption wavelengths, emission wavelengths, fluorescence lifetimes orother measurable optical property over the concentration range ofinterest is potentially measured using one of the indicators of thisinvention.

The indicator is generally prepared for use as a detection reagent bydissolving the indicator in solution at a concentration that is optimalfor detection of the indicator at the expected concentration of thetarget ion. Modifications that are designed to enhance permeability ofthe indicator through the membranes of living cells, such asacetoxymethyl esters and acetates, may require the indicator to bepredissolved in an organic solvent such as dimethylsulfoxide (DMSO)before addition to a cell suspension, where the indicators then readilyenter the cells. Intracellular enzymes cleave the esters to the morepolar acids and phenols that are then well retained inside the cells.For applications where permeability of cell-membranes is required, theindicators of the invention are typically substituted by only onefluorophore.

Therefore, a method for binding and detecting target ions in a live cellcomprises the following steps:

-   -   a) contacting a sample of live cells with a present compound;    -   b) incubating the sample and the compound for sufficient time to        allow the compound to chelate the target metal ion; and,    -   c) illuminating the sample with an appropriate wavelength to        generate a detectable fluorescent signal whereby the target ion        is detected in a live cell.

A specific indicator of the present invention is useful for thedetection and/or quantification of a desired target ion, when thebinding of the target ion in the metal ion-binding moiety of theindicator results in a detectable change in spectral properties.Preferably, the change in spectral properties is a detectablefluorescence response.

The optical response of the indicating reagent is determined by changesin absorbance or fluorescence, preferably fluorescence. If absorbancemeasurements are used to determine ion concentrations, then it isusually optimal to adjust the optical density of the indicator in thesample over the range of analyte concentration to a value ofapproximately 0.02 to 2.5 (most preferably 0.1 to 1). For fluorescencemeasurements, the concentration of the indicator will depend mostly onthe sensitivity of the equipment used for its detection.

If the optical response of the indicator will be determined usingfluorescence measurements, samples are typically stained with indicatorconcentrations of 10⁻⁹ M to 10⁻² M. The most useful range of analyteconcentration is about one log unit above and below the dissociationconstant of the ion-indicator complex. This dissociation constant isdetermined by titration of the indicator with a known concentration ofthe target ion, usually over the range of virtually zero concentrationto approximately 100 millimolar of the target ion, depending on whichion is to be measured and which indicator is being used. Thedissociation constant may be affected by the presence of other ions,particularly ions that have similar ionic radii and charge. It may alsobe affected by other conditions such as ionic strength, pH, temperature,viscosity, presence of organic solvents and incorporation of the sensorin a membrane or polymeric matrix, or conjugation or binding of thesensor to a protein or other biological molecule. Any or all of theseeffects need to be taken into account when calibrating an indicator.

The indicator is combined with a sample in a way that will facilitatedetection of the target ion concentration in the sample. The sample isgenerally a representative cell population, fluid or liquid suspensionthat is known or suspected to contain the target ion. Representativesamples include intracellular fluids such as in blood cells, culturedcells, muscle tissue, neurons and the like; extracellular fluids inareas immediately outside of cells; in vesicles; in vascular tissue ofplants and animals; in biological fluids such as blood, saliva, andurine; in biological fermentation media; in environmental samples suchas water, soil, waste water and sea water; in industrial samples such aspharmaceuticals, foodstuffs and beverages; and in chemical reactors.

In one embodiment of the invention, the sample contains cells, and theindicator is combined with the sample in such a way that the indicatoris present within the sample cells. By selection of the appropriatechelating moiety, fluorophore, and the substituents thereon, indicatorsare prepared that will selectively localize in desired organelles, andprovide measurements of the target ion in those organelles. Conjugatesof the indicators of the invention with organelle-targeting peptides areused to localize the indicator to the selected organelle, facilitatingmeasurement of target ion presence or concentration within the organelle(as described in U.S. Pat. No. 5,773,227). Alternatively, selection of alipophilic fluorophore, or a fluorophore having predominantly lipophilicsubstituents, wherein the lipophilic substituents are not labile, willresult in localization in lipophilic environments in the cell, such ascell membranes.

The metal-ion chelating compound is combined with a sample in a way thatwill facilitate detection of the target ion concentration in the sample.The sample is generally a representative cell population, fluid orliquid suspension that is known or suspected to contain the target ion.Representative samples include intracellular fluids such as in bloodcells, cultured cells, muscle tissue, neurons and the like;extracellular fluids in areas immediately outside of cells; in vesicles;in vascular tissue of plants and animals; in biological fluids such asblood, saliva, and urine; in biological fermentation media; inenvironmental samples such as water, soil, waste water and sea water; inindustrial samples such as pharmaceuticals, foodstuffs and beverages;and in chemical reactors. Detection and quantitation of the target ionin a sample can help characterize the identity of an unknown sample, orfacilitate quality control of a sample of known origin.

The sample can be a biological fluid such as whole blood, plasma, serum,nasal secretions, sputum, saliva, urine, sweat, transdermal exudates,cerebrospinal fluid, or the like. Biological fluids also include tissueand cell culture medium wherein zinc ions have been secreted into themedium. Alternatively, the sample may be whole organs, tissue or cellsfrom the animal. Examples of sources of such samples include muscle,eye, skin, gonads, lymph nodes, heart, brain, lung, liver, kidney,spleen, thymus, pancreas, solid tumors, macrophages, mammary glands,mesothelium, and the like. Cells include without limitation prokaryoticcells and eukaryotic cells that include primary cultures andimmortalized cell lines. Eukaryotic cells include without limitationovary cells, epithelial cells, circulating immune cells, β cells,hepatocytes, and neurons. Calcium and zinc ions have been determined toplay a role in many mammalian, if not all, organs and cell types suchthat there is no intended limitation on the sample for the presentinvention.

The end user will determine the choice of the sample and the way inwhich the sample is prepared. For example the sample may include amixture of cells, or prepared for HTS, or for imaging by flow cytometry.The sample includes, without limitation, any biological derived materialthat is thought to contain target ions, preferably calcium.Alternatively, samples also include material that target ions have beenadded to determine the effect the ions have on predetermined biologicalparameters.

A preferred embodiment of the present invention involves long wavelengthintracellular ion indicator. The long wavelength indicators avoidcellular autofluorescence and interference from other agents such asdrugs which have been introduced to cell. The long wavelengthintracellular ion indicators also allow for unique detection signals inmultiplexing applications, such as with GFP's or other indicators.Accordingly, one embodiment of the present invention involves detectionof multiple analytes or metal ions simultaneously via introduction ofthe compositions described herein and different indicator comprising areporter molecule or dye.

Quantification of target ion levels in samples is typically accomplishedusing the indicators of the present invention by methods known in theart. For example, the ratiometric measurement of ion concentrationprovides accurate measurement of ion concentrations by the treatment ofthe fluorescence data as the ratio of excitation or fluorescenceintensities at two wavelengths, rather than the absolute intensity at asingle wavelength. Using the ratio method, a number of variables thatmay perturb the ion concentration measurements are eliminated. Inparticular, ion-dependent factors that affect the signal intensity, suchas nonuniform intracellular dye concentrations, probe leakage, dyebleaching and cell thickness, are canceled in the ratio measurements,since these parameters have a similar effect on intensities at bothwavelengths. While the ratio method can be used to determineconcentrations using observation of either the excitation spectra of theindicator, the emission spectra of the indicator, or both, in the caseof the indicators of the present invention, the shift in excitationenergy upon binding metal ions makes observation of the excitationspectrum a more useful technique. In either case, to achieve maximalutility, the indicator must be calibrated (to compensate for variance inthe dissociation constant of the indicator due to ionic strength,viscosity, or other conditions within the sample). To calibrate theindicator, ionophores such as A-23187, gramicidin, valinomycin, orionomycin are used. Non-ratiometric analysis can also be accomplished bycalibration with a second fluorescent dye present in the sample.

The optical response of the indicator to the ion can be detected byvarious means that include measuring absorbance or fluorescence changeswith an instrument, visually, or by use of a fluorescence sensingdevice. Several examples of fluorescence sensing devices are known, suchas fluorometers, fluorescence microscopes, laser scanners, flowcytometers, and microfluidic devices, as well as by cameras and otherimaging equipment.

Kits of the Invention

Due to the advantageous properties and the simplicity of use of thepresent intracellular ion indicators, they are particularly useful inthe formulation of a kit for the complexing, detection, quantificationor monitoring of selected target ions, such as calcium, comprising oneor more compounds or compositions of the invention in any of theembodiments described above (optionally in a stock solution),instructions for the use of the compound to complex or detect a desiredtarget ion, and optionally comprising additional components.

A particular kit for binding an intracellular metal ion in a sample,comprises:

a compound of formula:G_(L)-DYE-M_(c)

wherein,

G_(L) is a lipophilic group:

DYE is a fluorescent dye; and

M_(c) is a metal chelating moiety; and

one or more components selected from the group consisting of acalibration standard of an intracellular metal ion, an ionophore, afluorescent standard, an aqueous buffer solution and an organic solvent.

A kit of the present invention for binding a target metal ion in asample may comprise a compound as described herein and instructions foruse thereof. The kit may further comprise one or more componentsselected from the group consisting of a calibration standard of a metalion, an ionophore, a fluorescent standard, an aqueous buffer solution,control cells and an organic solvent.

The additional kit components may be selected from, without limitation,calibration standards of a target ion, ionophores, fluorescencestandards, aqueous buffers, control cells and organic solvents. Theadditional kit components are present as pure compositions, or asaqueous solutions that incorporate one or more additional kitcomponents. Any or all of the kit components optionally further comprisebuffers.

Illumination:

In a typical detection method, at any time after or during binding ofthe compounds of the present invention with the target metal ion, thesample is visualized whereby the compound is detected. Visualization cancomprise different methods and is dependent on the reporter moleculethat is covalently attached to the metal ion chelator. When the reportermolecule is a dye label, visualization typically comprises illuminationwith a wavelength of light capable of exciting the dye to produce adetectable optical response, as defined above, and observed with a meansfor detecting the optical response. Equipment that is useful forilluminating the dye compounds of the invention includes, but is notlimited to, hand-held ultraviolet lamps, mercury arc lamps, xenon lamps,lasers and laser diodes. These illumination sources are optionallyintegrated into laser scanners, fluorescence-based microplate readers,standard or minifluorometers, flow cytometers or chromatographicdetectors. The degree and/or location of binding/chelation, comparedwith a standard or expected response, indicates whether and to whatdegree the sample possesses a given characteristic, i.e., cellprocesses/activity.

The optical response is optionally detected by visual inspection, or byuse of any of the following devices: CCD cameras, video cameras,photographic film, laser-scanning devices, fluorometers, photodiodes,quantum counters, epifluorescence microscopes, scanning microscopes,fluorescence-based microplate readers, or by a means for amplifying thesignal such as photomultiplier tubes.

Thus, it is contemplated by the present invention that a wide variety ofinstrumentation may be used to detect target metal ions.

As described above, while a wide variety of methods of detection areused with the present invention, a preferred method includes the use offluorescence. Fluorescence from the compound binding to the target metalion can be visualized with a variety of imaging techniques, includingordinary light or fluorescence microscopy.

The examples below are given so as to illustrate the practice of thisinvention. They are not intended to limit or define the entire scope ofthis invention.

EXAMPLES

The present invention further provides compounds, intermediates, methodsof synthesis and methods of use involving any of the following examplecompounds.

The starting materials for the synthesis of calcium ion indicators weredescribed in: Martin, V. V.; Beierlein, M.; Morgan, J. L.; Rothe, A.;Gee, K. R. Cell Calcium 2004, 36, 509, and in: Beierlein, M.; Gee, K.R.; Martin, V. V.; Regehr, W. G. J. Neurophysiol. 2004, 92(1), 591.Preparation of the starting materials for sodium indicators werepublished in: Martin, V. V.; Rothe, A.; Diwu, Z.; Gee, K. R. Bioorg.Med. Chem. Lett. 2004, 14, 5313, and in Martin, V. V.; Rothe, A.; Gee,K. R. Bioorg. Med. Chem. Lett. 2005, 15, 1855.

Example 1

Synthesis of a cell-permeable calcium ion indicator 146 havingtetrakis(2-carboxyethyl)rhodamine fluorophore [synthesis of thisfluorophore was adopted from: Bandichhor, R.; Petrescu, A. D.; Vespa,A.; Kier, A. B.; Schroeder, F.; Burgess, K. Bioconjugate Chem. 200617(5), 1219] with extra four carboxylic groups protected as AM esters.(Scheme 100).

Compound 143. The mixture of the aldehyde 141 (1.120 g, 2 mmol), diester142 (1.348 g, 4.8 mmol) and 10-camphorsulfonic acid (40 mg; catalyst) inEtCOOH (40 mL) was stirred at 70° C. for 48 h. It was cooled to rt,poured into a mixture of 3N NaOAc (500 mL) and sat. NaHCO₃ 200 mL), andextracted with CHCl₃ (200 mL+8×50 mL). The extract was washed with satNaCl (300 mL), dried over MgSO₄ to give the compound 143 (2.20 g,˜100%), which was used in the next step without purification.

Octamethyl ester 144. A crude dihydro compound 143 (˜2 mmol) was stirredwith chloranil (0.984 g, 4 mmol) in CHCl₃/MeOH (1:1) (60 mL) for 5 h.The mixture was filtered, evaporated. The residue was re-dissolved inCHCl₃ (25 mL), loaded onto silica gel column (6×50 cm bed, packed in 3%MeOH and 1% AcOH in CHCl₃) and chromatographed in 3% to 10% gradientMeOH in CHCl₃ and 1% AcOH. The fractions, containing product wereevaporated, co-evaporated with toluene (50 mL), dissolved in CHCl₃ (250mL), allowed to stand for 3 h, filtered from silicates and evaporated togive the compound 144 (0.647 g, 28%) as a dark red oil.

Heptapotassium salt 145. To a stirred solution of the compound 144 (55mg, 0.02 mmol) in MeOH (2 mL) was added 1 N KOH (0.5 mL, 0.5 mmol). Themixture was stirred for 16 h, diluted with H₂O (5 ml) and 0.2 N HCl wasadded to pH 9.5. The solution was evaporated, the residue wasre-dissolved in H₂O (3 mL) and loaded onto Sephadex LH-20 column (2.6×95cm bed, packed with H₂O) and eluted with H₂O. Combined fractionscontaining the product were evaporated to ˜2 mL volume and lyophilizedto give the compound 145 (8 mg, 43%) as hygroscopic dark red flakes.

Octo-AM ester 146. To a solution of the salt 145 (2.5 mg, 0.002 mmol) inMeOH (2 mL) a dry tetrabutylammonium hydrosulfate (17 mg, 0.05 mmol) wasadded. The mixture was stirred for 5 min, and evaporated. The residuewas dissolved in DMF (0.2 mL); DIEA (70 μL, 0.4 mmol) was added followedby bromomethyl acetate (20 μL, 0.2 mmol). The resulted solution wasstirred for 16 h and diluted with CHCl₃ (50 mL). Chloroform solution waswashed with 1% AcOH (3×30 mL), H₂O (2×30 mL), brine (50 mL), filteredthrough paper and evaporated to give the compound 146 (3.0 mg, 91%) as adark red oil.

Example 2

Synthesis of the compounds 153 and 154, reduced-leakage sodium ionindicators for extracellular and intracellular applications (scheme 101)

Compound 148. To a stirred solution of the diamine 147 (1.690 g, 5 mmol)in THF (20 mL) and DIEA (2.6 mL), methoxyacetyl chloride (1.1 mL, 12mmol) in THF (20 mL) was added dropwise while cooling on the ice bath.The mixture was for 15 min, then cooling was removed and the stirringwas continued for 2 h. The DIEA salt was filtered off; filtrate wasevaporated, the residue was dissolved in EtOAc (200 mL), washed with 1%HCl (2×100 mL), H₂O (100 mL), brine (100 mL), filtered through paperfilter and evaporated to give the compound 148 (1.71 g, 00) as anoff-white foamy solid.

Compound 149. Bis-acyl derivative 148 (1.70 g, 3.73 mmol) and B₂H₆-THFcomplex (1 N in THF; 37 mL, 37 mmol) in THF (50 mL) were refluxedovernight, cooled to rt and carefully decomposed with methanol (100 mLtotal) [warning: controlled addition required; violent reaction!]. Themixture was again heated to reflux, stirred for more 1 h, evaporated,and co-evaporated with MeOH (3×50 mL) to give the compound 149 (1.585 g,99%) as a yellowish viscous oil.

Aldehyde 150. A mixture of the compound 149 (1.580 g, 3.69 mmol) and(chloromethylene)dimethylammonium chloride (2.36 g, 18.46 mmol) in DMF(20 mL) was stirred at 50° C. for 48 h, cooled to rt and poured intoice/sat. NaHCO₃ (1:1, 200 mL). The mixture was extracted with CHCl₃(10×50 mL), the extract was washed with brine (100 mL), dried overMgSO₄, and evaporated. The residue was purified by column chromatographyon a silica gel column (packed in 3% MeOH in CHCl₃) using 3% MeOH inCHCl₃ as eluant to give the aldehyde 150 (0.425 g, 24%) as a yellowishoil.

Compound 151. The mixture of the aldehyde 150 (0.420 g, 0.84 mmol),diester 142 (0.566 g, 2.02 mmol) and 10-camphorsulfonic acid (18 mg;catalyst) in EtCOOH (17 mL) was stirred at 70° C. for 24 h. It wascooled to rt, poured into a mixture of 3N NaOAc (200 mL) and 1 N NaOH (5mL), and extracted with CHCl₃ (100 mL+6×30 mL). The extract was washedwith sat NaCl (300 mL), dried over MgSO₄ to give the compound 151 (0.8g, ˜100%), which was used in the next step without purification.

Tetramethyl ester 152. A crude dihydro compound 151 (˜0.8 mmol) wasstirred with chloranil (0.590 g, 4 mmol) in CHCl₃/MeOH (1:1) (3 mL) for5 h. The mixture was filtered, evaporated. The residue was re-dissolvedin CHCl₃ (25 mL), loaded onto silica gel column (3×50 cm bed, packed in5% MeOH and 1% AcOH in CHCl₃) and chromatographed in 5% to 10% gradientMeOH in CHCl₃ and 1% AcOH. The fractions, containing product wereevaporated, co-evaporated with toluene (50 mL), dissolved in CHCl₃ (150mL), allowed to stand for 3 h, filtered from silicates and evaporated togive the compound 152 (0.147 g, 18%) as a dark red oil.

Tetra (tetramethylammonium) salt 153. To a stirred solution of thecompound 152 (21 mg, 0.02 mmol) in MeOH (1 mL) and dioxane (1 mL) wasadded tetramethylammonium hydroxide (−2.8 N in water; 0.15 mL, 0.4mmol). The mixture was stirred for 20 h and evaporated; the residue wasre-dissolved in H₂O (3 mL) and loaded onto Sephadex LH-20 column (2.6×95cm bed, packed with H₂O) and eluted with H₂O. Combined fractionscontaining the product were evaporated to ˜2 mL volume and lyophilizedto give the compound 153 (22 mg, 95%) as hygroscopic dark red flakes.

Tetra-AM ester 154. To a solution of the salt 153 (11 mg, 0.01 mmol) inDMF (0.1 mL); DIEA (174 μL, 1 mmol) was added followed by bromomethylacetate (23 μL, 0.25 mmol). The resulted solution was stirred for 4 hand diluted with CHCl₃ (50 mL). Chloroform solution was washed with 1%AcOH (3×30 mL), H₂O (2×30 mL), dried over MgSO₄, filtered and evaporatedto give the compound 154 (10 mg, 79%) as a dark red hygroscopic solid.

Example 3

Synthesis of the compounds 156 and 157: reduced-leakage na⁺indicators—for extracellular and intracellular environments

Dicarboxylic acid 156. To a stirred solution of the compound 155 (114mg, 0.2 mmol) in MeOH (2 mL), is added iminodiacetic acid (33 mg, 0.25mmol) in 6N KOH (1 mL). Diluted hydrochloric acid (0.2 N) is added to pH9.5 and the solution of 37% formaldehyde (0.8 mL, 1 mmol) in MeOH (2 mL)is introduced. The mixture is stirred for 10 h, and evaporated. Theresidue is dissolved in water (100 mL), washed with CHCl₃ (50 mL),acidified with 0.2 N HCl to pH 4, and extracted with CHCl₃ (10×20 mL).Extract is dried over MdSO₄, and evaporated. The residue is loaded ontochromatography column (2.5×40 cm bed, packed with CHCl₃) and eluted with3% to 10% MeOH gradient in CHCl₃ to give the compound 156.

Tris-AM derivative 157. To a solution of the dicarboxylic acid 156 (36mg, 0.05 mmol) in DMF (2 mL); DIEA (175 μL, 1.0 mmol) is added followedby bromomethyl acetate (50 μL, 0.53 mmol). The resulted solution isstirred for 6 h and diluted with CHCl₃ (100 mL). Chloroform solution waswashed with H₂O (5×50 mL), brine (50 mL), filtered through paper filterand evaporated to give the compound 157.

Example 4

Spectroscopic study of the calcium binding to compound 145 (λ_(ex)=555nm, λ_(em)=580 nm; K_(d)=380 nmol)

-   a) Changes in emission spectrum upon increase of [Ca²⁺]    concentration    -   b) Determination of the binding constant. Results are shown in        FIG. 1.

Example 5

Imaging of cellular localization of a) conventional red Ca indicatorRhod-2 (Mitochondrial Localization), b) Compound 146, and c) CytosolicIndicator Fluo-4

Results are shown in FIG. 2.

Example 105

Determination of intracellular calcium with Compound 146 in a MultiwellPlate

CHOM1 cells were plated in poly d lysine coated 96 well plates at adensity of 35,000 cells per well on the morning of the experiment andallowed to settle for four hours in complete medium. The medium wasaspirated from the cells and 100 uL HBSS buffer was added. The HBSSbuffer was prepared by adding 20 mM HEPES to calcium and magnesiumcontaining HBSS buffer (IVGN catalogue number 14025) and pH set to 7.4with NaOH. This solution was filter sterilized and stored at 4 C.Following incubation the HBSS buffer was aspirated and replace with 100uL Loading Buffer. The Loading Buffer (10 ml for one 96 well plate) wasprepared as follows: To a 15 mL tube, add in order

100 uL Powerload (IVGN catalogue number F10017); 10 uL of 10 mM dye inDMSO

Mix gently by swirling (Stock preparations of Compound 146 were preparedat 10 mM in anhydrous DMSO and aliquots stored frozen at −20 C) and 10mL Probenecid containing Assay Buffer. The assay buffer was prepared bydiluting water soluble probenecid stock 1:100 in HBSS buffer on the dayof use. The water soluble probenecid (Invitrogen catalogue numberP36400) was prepared at 100× by adding 1 mL deionized water to a 77 mgvial of water soluble probenecid. Unused portions were stored frozen at−20 C.

Following incubation with Loading Buffer the cells are placed 37 C for60 minutes room atmosphere, not 5% CO2 incubator. The loading buffer wasaspirated and replaced with 100 uL Probenecid containing Assay Buffer.The Loading buffer was then replaced with 100 uL Probenecid containingAssay Buffer

The cells are imaged using a standard microplate reader(FlexStationII384, Molecular devices, Sunnyvale Calif.) set to 550nanometers (nm) excitation, 600 nm emission with excitation cutoff at590 nm. Readings are taken every 2 seconds for 20 seconds and then 25 uLof carbachol containing stimulus is added to the microwells for a 1:5dilution. A 1 uM final carbachol stimulation results in a signal such asseen in FIG. 3A.

A dose-response study of carbachol in CHOM1 cells loaded as above with10 and 20 uM Rhod-2 vs 10 and 20 uM Compound 146 is shown in FIG. 2.

1. An intracellular ion indicator compound, wherein the compoundcomprises a metal chelating moiety (M_(c)), a reporter molecule and twoor more lipophilic groups (GO covalently bonded through a linker to thereporter molecule, wherein the lipophilic groups, when present in a livecell, are cleaved resulting in two or more negatively charged groups. 2.The compound of claim 1, wherein the metal chelating moiety (M_(c)) iscovalently bonded through a PET linker to the reporter molecule.
 3. Thecompound of claim 1, wherein the reporter molecule is a fluorescent dye.4. The compound of claim 1, wherein the lipophilic groups comprise anester.
 5. The compound of claim 4, wherein the two or more negativelycharged groups are carboxylate groups.
 6. The compound of claim 1,wherein the lipophilic groups are selected from the group consisting ofC₁-C₆ carboxyalkyl, —(CH₂)₁₋₆COOCR¹⁵ ₂OC(═O)(CH₂)₀₋₄CH₃, andalpha-acyloxyalkyl; wherein R¹⁵ is H, alkyl or substituted alkyl.
 7. Thecompound of claim 1, wherein the lipophilic groups are:—CH₂CH₂COOCH₂OC(═O)CH₃.
 8. The compound of claim 3, wherein thefluorescent dye comprises at least three lipophilic groups.
 9. Thecompound according to claim 3, wherein the compound has the formula:(G_(L))_(v)-L-DYE-M_(c) wherein, L is a linker; G_(L) is a lipophilicgroup; DYE is a fluorescent dye; M_(c) is a metal chelating moiety; andv is 2, 3, 4, or
 5. 10. The compound of claim 9, wherein DYE is a3-aminoxanthene or tautomer thereof.
 11. The compound of claim 1,wherein M_(c) is a metal ion chelator capable of binding calcium,magnesium, zinc, iron, thallium, sodium or potassium.
 12. The compoundof claim 1, wherein M_(c) comprises BAPTA or BAPTA derivative, a crownether moiety, a cryptan, APTRA, FluoZin-1, 2 or 3 or a phenanthroline.13. The compound of claim 1, wherein M_(c) has the structure:

wherein, R¹, R², R³ and R⁴ are each independently selected from thegroup consisting of carbonyl, substituted carbonyl, carboxyl, alkyl,substituted alkyl, and -L-(G_(L))_(w); or R² and R⁴ are bound togetherby: —(CH₂CH₂—O)_(t)—CH₂CH₂—, wherein t is 1 or 2; R⁵ and R⁶ are eachindependently selected from the group consisting of -L-(G_(L))_(w),alkyl, substituted alkyl, carbonyl, substituted carbonyl, alkoxy,substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino,aminocarbonyl, aminothiocarbonyl, aminocarbonylamino,aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl,aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester,(carboxyl ester)amino, (carboxyl ester)oxy, cyano, halo, hydroxy, nitro,SO₃ ⁻, sulfonyl, substituted sulfonyl, sulfonyloxy, thioacyl, thiol,alkylthio, substituted alkylthio, aryl, substituted aryl, heteroaryl,substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl; L is a linker; G_(L) is alipophilic group; w is 1 or 2; and m and n are each independently 0, 1,2, or
 3. 14. The compound of claim 13, wherein n is 1 and R⁵ is —CH₃.15. The compound of claim 13, wherein R² and R⁴ are bound together by:—CH₂CH₂—O—CH₂CH₂—.
 16. The compound of claim 13, wherein R¹, R², R³ andR⁴ are -L-(G_(L))_(w).
 17. The compound of claim 13, wherein L is asingle covalent bond, or a covalent linkage that is linear or branched,cyclic or heterocyclic, saturated or unsaturated, having 1-30nonhydrogen atoms selected from the group consisting of C, N, P, 0 andS; and are composed of any combination of ether, thioether, amine,ester, carboxamide, sulfonamide, hydrazide bonds and aromatic orheteroaromatic bonds.
 18. The compound of claim 13, wherein L is -oxo-,alkoxy, -amino-, or -substituted amino-.
 19. The compound of claim 1,wherein M_(c) has the structure:

wherein, X is —(CH₂CH₂—O)_(y)—CH₂CH₂—, wherein y is 1, 2, 3, 4, or 5; R¹is H, alkyl, substituted alkyl, carbonyl, or substituted carbonyl; R⁶ isselected from the group consisting of -L-(G_(L))_(w), alkyl, substitutedalkyl, carbonyl, substituted carbonyl, alkoxy, substituted alkoxy, acyl,acylamino, acyloxy, amino, substituted amino, aminocarbonyl,aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino,aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino,amidino, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxylester)oxy, cyano, halo, hydroxy, nitro, SO₃ ⁻, sulfonyl, substitutedsulfonyl, sulfonyloxy, thioacyl, thiol, alkylthio, substitutedalkylthio, aryl, substituted aryl, heteroaryl, substituted heteroaryl,cycloalkyl, substituted cycloalkyl, heterocyclyl, and substitutedheterocyclyl; L is a linker; w is 1 or 2; G_(L) is a lipophilic group;and m is 0, 1, 2, or
 3. 20. The compound of claim 3, wherein thefluorescent dye comprises a 3-aminoxanthene or a tautomer thereof. 21.The compound of claim 3, wherein the fluorescent dye comprises a3,6-diaminoxanthene or a tautomer thereof.
 22. The compound of claim 3,wherein the fluorescent dye has the structure:

wherein, R⁹ and R¹⁰ are each independently selected from the groupconsisting of alkyl, substituted alkyl, alkoxy, substituted alkoxy,acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl,aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino,aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino,amidino, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxylester)oxy, cyano, halo, hydroxy, nitro, SO₃, sulfonyl, substitutedsulfonyl, sulfonyloxy, thioacyl, thiol, alkylthio, substitutedalkylthio, aryl, substituted aryl, heteroaryl, substituted heteroaryl,cycloalkyl, substituted cycloalkyl, heterocyclyl, substitutedheterocyclyl, and -L-(G_(L))_(w); R⁷ is ═O or ═N(R¹¹)₂, wherein R¹¹ isH, alkyl, (G_(L))_(w), substituted alkyl, carbonyl, or substitutedcarbonyl; R⁸ is hydroxyl, —OR₁₃ or —N(R¹²)₂ wherein R¹² and R¹³ areindependently H, alkyl, (G_(L))_(w), substituted alkyl, carbonyl, orsubstituted carbonyl; or one or both of R⁸ and (R¹⁰)_(p) and R⁷ and(R⁹)_(q) are taken together to form a fused aryl or heteroaryl group; Lis a linker; G_(L) is a lipophilic group; w is 1 or 2; and p and q areeach independently 0, 1 or 2; wherein the dye comprises at least twoG_(L) groups.
 23. The compound of claim 22, wherein R⁷ is ═O, R⁸ ishydroxyl, p+q=1 and R⁹ or R¹⁰ is -L-(G_(L))_(w).
 24. The compound ofclaim 22, wherein R⁹ is -L-(G_(L))_(w) and q is
 1. 25. The compound ofclaim 22, wherein L is a single covalent bond, or a covalent linkagethat is linear or branched, cyclic or heterocyclic, saturated orunsaturated, having 1-30 nonhydrogen atoms selected from the groupconsisting of C, N, P, O and S; and are composed of any combination ofether, thioether, amine, ester, carboxamide, sulfonamide, hydrazidebonds and aromatic or heteroaromatic bonds.
 26. The compound of claim22, wherein L is alkyl, substituted alkyl, -oxo-, alkoxy, -amino-, or-substituted amino-.
 27. The compound of claim 22, wherein R⁷ is═N⁺(G_(L))₂ and R⁸ is —OG_(L).
 28. The compound of claim 22, werein R⁷is ═N⁺(G_(L))₂ and R⁸ is —N(G_(L))₂.
 29. The compound of claim 28,wherein G_(L) is —(CH₂)₁₋₆COOCR¹⁵ ₂OC(═O)(CH₂)₀₋₄CH₃ wherein R¹⁵ is H,alkyl or substituted alkyl.
 30. The compound of claim 28, wherein G_(L)is —CH₂CH₂COOCH₂OC(═O)CH₃.
 31. The compound of claim 3, wherein thefluorescent dye has the structure:

R⁹ and R¹⁰ are each independently selected from the group consisting ofalkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino,acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl,aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy,aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl,carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, halo,hydroxy, nitro, SO₃ ⁻, sulfonyl, substituted sulfonyl, sulfonyloxy,thioacyl, thiol, alkylthio, substituted alkylthio, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, cycloalkyl, substitutedcycloalkyl, heterocyclyl, substituted heterocyclyl and -L-(G_(L))_(w); Lis a linker; G_(L) is a lipophilic group; w is 1 or 2; and p and q areeach independently 0, 1 or
 2. 32. The compound of claim 31, whereinG_(L) comprises an ester.
 33. The compound of claim 31, wherein G_(L) isselected from the group consisting of C₁-C₆ carboxyalkyl,—(CH₂)₁₋₆COOCR¹⁵ ₂OC(═O)(CH₂)₀₋₄CH₃, and alpha-acyloxyalkyl; wherein R¹⁵is H, alkyl or substituted alkyl.
 34. The compound of claim 31, whereinthe G_(L) is: —CH₂CH₂COOCH₂OC(═O)CH₃.
 35. A method for binding aintracellular metal ion in a sample, comprising: contacting the samplewith a intracellular ion indicator compound, wherein the compoundcomprises a metal chelating moiety, a reporter molecule and one or morelipophilic groups covalently bonded to the reporter molecule through alinker wherein the lipophilic groups, when present in a live cell, arecleaved resulting in one or more negatively charged groups; andincubating the sample and compound for sufficient time to allow thecompound to chelate the intracellular ion whereby the intracellular ionis bound.
 36. The method of claim 35, wherein the method furthercomprises illuminating the compound with a suitable light source wherebythe calcium ion is detected.
 37. The method of claim 35, wherein thesample comprises cells.
 38. The method of claim 37, wherein theincubating step comprises incubating the sample and compound forsufficient time to allow the compound to enter a cell.
 39. The method ofclaim 38, wherein the intracellular ion is bound in the cell.
 40. Amethod for identifying intracellular intracellular metal ions in asample, comprising: contacting the sample with a intracellular ionindicator compound, wherein the compound comprises a metal chelatingmoiety, a reporter molecule and one or more lipophilic groups covalentlybonded to the reporter molecule through a linker wherein the lipophilicgroups, when present in a live cell, are cleaved resulting in one ormore negatively charged groups; incubating the sample for a period oftime sufficient for the compound to enter the cell; cleaving the one ormore lipophilic groups; and illuminating the sample with an appropriatewavelength, whereby the intracellular ions are identified.
 41. Acomposition comprising an intracellular metal ion and an intracellularion indicator compound, wherein the compound comprises a metal chelatingmoiety (M_(c)), a reporter molecule and two or more lipophilic groups(G_(l)) covalently bonded through a linker to the reporter molecule,wherein the lipophilic groups, when present in a live cell, are cleavedresulting in two or more negatively charged groups.
 42. A kit forbinding an intracellular metal ion in a sample, comprising: aintracellular ion indicator compound, wherein the compound comprises ametal chelating moiety (M_(c)), a reporter molecule and two or morelipophilic groups (G_(L)) covalently bonded through a linker to thereporter molecule, wherein the lipophilic groups, when present in a livecell, are cleaved resulting in two or more negatively charged groups;and one or more components selected from the group consisting of acalibration standard of the intracellular metal ion, an ionophore, afluorescent standard, an aqueous buffer solution and an organic solvent.43. A method of making a cell permeable compound, wherein the compoundcomprises a metal chelating moiety (M_(c)), a reporter molecule and twoor more lipophilic groups (G_(L)) covalently bonded through a linker tothe reporter molecule, wherein the lipophilic groups, when present in alive cell, are cleaved resulting in two or more negatively chargedgroups, the method comprising: contacting the metal ion chelator (M_(c))with the reporter molecule, such that the reporter molecule covalentlybinds to the metal ion chelator; and contacting a lipophilic group witha negatively charged substituent present on the reporter molecule, suchthat the lipophilic group binds to the negatively charged group therebyneutralizing the negatively charged group.
 44. A compound selected fromthe group consisting of:

wherein AM is —CH₂OC(═O)CH₃.
 45. A compound selected from the groupconsisting of:

wherein h and i are independently 0-4; z is 0 or 1; and AM is—CH₂OC(═O)CH₃.